DESCRIPTION METHODS AND COMPOSITIONS FOR A DEUBIOUITINATING ENZYME AND VARIANTS THEREOF BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is generally directed to crystal protein structures, and more specifically to Ubiquitin C-terminal hydrolase, which catalyzes the removal of adducts from the C-terminus of ubiquitin.

2. Description of Related Art Ubiquitin is a small (8. 6 kDa) highly conserved protein that is best known for its role in targeting proteins for degradation by the 26S protease. Recent reviews include (Ciechanover and Schwartz, 1994 ; Hershko and Ciechanover, 1992 ; Jentsch, 1992 ; Wilkinson et al., 1995). Ubiquitin has been implicated in numerous cellular processes, including : cell cycle control, oncoprotein degradation, receptor function, apoptosis, regulation of transcription, stress responses, maintenance of chromatin structure, DNA repair, signaling pathways, antigen presentation, and the degradation of abnormal proteins. Monomeric ubiquitin is activated by E1 (ubiquitin activating enzyme), which forms a thiolester bond with the ubiquitin C-terminus. Families of E2 (ubiquitin conjugating) and E3 (ubiquitin ligase) enzymes then catalyze ligation of the ubiquitin C-terminus to lysine side chains of acceptor proteins. Acceptor proteins can be modified with a single ubiquitin attached to one or more different lysine side chains. Alternatively, acceptor proteins can be polyubiquitinated, with a lysine side chain of the first ubiquitin conjugated to the C-terminus of the next, to form long chains attached to the target protein. Efficient targeting for degradation by the 26S protease appears to require polyubiquitination (Chau et al., 1989 ; Gregori et al., 1990). In addition to targeting proteins for degradation by the 26S protease, other roles of ubiquitination include modification of chromatin structure (Bradbury, 1992)

lysosomal targeting (Hicke and Riezman, 1996) and regulation of a kinase activity (Chen et al., 1996).

In addition to isopeptide linkages to the lysine side chains of acceptor proteins, the ubiquitin C-terminus is also found attached to a-amino groups in peptide bonds, since all known ubiquitin genes encode fusion proteins in which ubiquitin is followed by a C-terminal extension (Ozkaynak et al., 1987). Proteolytic processing at the ubiquitin C-terminus is catalyzed by deubiquitinating enzymes (DUB). Such processing is likely to be required for several different functions, including : liberation of monomeric ubiquitin from the polyprotein precursors, release of polyubiquitin chains from the remnants of 26S protease substrates, disassembly of polyubiquitin chains to allow recycling of monomeric ubiquitin, reversal of regulatory ubiquitination, editing of inappropriately ubiquitinated proteins, and regeneration of active ubiquitin from adducts with small cellular nucleophiles (such as glutathione) that may be produced by side reactions. Additionally, several ubiquitin-like proteins that occur as fusions or conjugates have been identified, at least some of which appear to undergo a similar processing (Haas et al., 1996 ; Matunis et al., 1996 ; Narasimhan et al., 1996 ; Olvera and Wool, 1993).

In light of the many different substrates, and the extensive biological consequences of ubiquitination, it is not surprising that numerous deubiquitinating enzymes have been identified. These enzymes fall into two distinct families of cysteine proteases, UBPs (ubiquitin-specific proteases) (Baker et al., 1992 ; Tobias and Varshavsky, 1992) and UCHs (ubiquitin C-terminal hydrolases) (Pickart and Rose, 1985). Both classes of enzymes hydrolyze the peptide bond (either a-or s- linked) at the C-terminus of ubiquitin. The UBP enzymes, 16 of which have been identified in yeast, were named for their ability to cleave large model fusion proteins at the C-terminus of ubiquitin. They vary in molecular weight from 50 kDa to 300 kDa, and exhibit a broad range of substrate specificity. Roles assigned for UBPs include cleavage of ubiquitin from the remnants of degraded protein (Papa and Hochstrasser, 1993) and disassembly of polyubiquitin chains to yield functional

monomers (Wilkinson et al., 1995). They appear to function in cell fate determination (Huang et al., 1995), transcriptional silencing (Henchoz et al., 1996 ; Moazed and Johnson, 1996), and the response to cytokines (Zhu et al., 1996).

The well characterized UCH enzymes are generally smaller than the UBPs, (25-28 kDa), although two larger sequences have been deposited in the GenBank database. Disruption or deletion of the one UCH gene identified in yeast confers no discernible phenotype, suggesting that the substrate specificity of UCH enzymes may overlap with the UBP enzymes (Baker et al., 1992 ; Miller et al., 1989). Biochemical studies have demonstrated that the human enzymes, UCH-L1 and UCH-L3, and the UCHs from S. cerevisiae and D. melanogaster hydrolyze s-linked amide bonds at the C-terminus of ubiquitin (Cohen) (Roff et al., 1996 ; Wilkinson, 1997), although most studies have focused on the hydrolysis of a-linked peptide bonds and small thiolester, ester, and amide linked adducts (Pickart and Rose, 1986 ; Wilkinson et al., 1986). In general, most of these small adducts are good substrates, except for peptide extensions with proline immediately following the scissile bond. UCH-L3 cleaves peptide extensions of up to 20 residues from ubiquitin with high efficiency and low sequence preference, while larger folded extensions are not cleaved (Wilkinson, 1997). Similar results have been reported for the yeast UCH (Liu et al., 1989 ; Miller et al., 1989).

These data suggest that the UCH enzymes may function to regenerate active ubiquitin from adducts with small nucleophiles (Pickart and Rose, 1985). The observed tissue specificity of UCH enzymes may reflect a distinct sets of substrate (s) (Wilkinson et al., 1992). UCH-L1 is identical to PGP9. 5, the neuronal ubiquitin C-terminal hydrolase that constitutes several percent of the total soluble protein in mammalian brain (Wilkinson et al., 1989). UCH-L2 appears to be constitutively expressed in many tissues, while UCH-L3 is expressed in hematopoetic cells.

An alignment of five UCH sequences shows that only 12% of the residues are invariant (FIG. 1). Site directed mutagenesis of invariant residues on UCH-L1 implicates Cys-95 (UCH-L3 numbering) as the active site nucleophile, and His-169 as

the general base in catalysis, with an important role also played by Asp-184 (Larsen et al., 1996). The UCH enzymes do not appear to share significant sequence similarity with any other protein.

In order to understand better the catalytic mechanism and substrate specificity of UCH enzymes, the inventors have determined the crystal structure of recombinant human UCH-L3 at a resolution of 1. 8 A. This structure has some similarities with the papain family of cysteine proteases, including an active site catalytic triad and oxyanion hole. A major topological difference from papain includes a 20-residue disordered loop that spans the active site. Based upon the structure, the present invention sets forth a binding orientation for ubiquitin substrates on UCH enzymes.

Moreover, the invention shows that the UCH active site is normally closed and opens upon binding to substrate, and that the disordered loop may function to define the substrate specificity of UCH enzymes.

SUMMARY OF THE INVENTION In one aspect, the present invention provides an isolated and purified amino acid sequence that encodes a deubiquitinating enzyme polypeptide UCH-L3. Preferably, a UCH-L3 peptide of the invention is a synthetic or recombinant polypeptide. More preferably, a polynucleotide of the present invention encodes a polypeptide comprising the structure of FIG. 1.

In certain embodiments, an amino acid sequence of the present invention encodes a variant UCH-L3 molecule that possesses structural differences from the native UCH-L3 protein. Such structural differences include greater stability ; i. e. ability to resist the effects of oxidation, heat, and so forth. Moreover, such structural differences may include UCH-L3 variants that are capable of cleaving larger proteins from the ubiquitin molecule than may be accomplished by the native UCH-L3 protein.

A further advantage of the present invention includes the production of inhibitors of UCH-L3 proteins that specifically interact at the active site to reduce or eliminate UCH-L3 activity.

In yet another embodiment, the present invention contemplates a process of preparing an UCH-L3 or variant UCH-L3 comprising transfecting a cell with polynucleotide that encodes an UCH-L3 or variant UCH-L3 polypeptide to produce a transformed host cell ; and maintaining the transformed host cell under biological conditions sufficient for expression of the polypeptide. The transformed host cell can be a eukaryotic cell. Alternatively, the host cell is a prokaryotic cell.

In still another embodiment, the present invention provides an antibody immunoreactive with an UCH-L3 or variant UCH-L3. Preferably, an antibody of the invention is a monoclonal antibody.

In another aspect, the present invention contemplates a process of producing an antibody immunoreactive with an UCH-L3 or variant UCH-L3 comprising the steps of (a) transfecting a recombinant host cell with a polynucleotide that encodes an UCH-L3 or variant UCH-L3 ; (b) culturing the host cell under conditions sufficient for expression of the polypeptide ; (c) recovering the polypeptide ; and (d) preparing the antibody to the polypeptide.

In yet another aspect, the present invention contemplates a process of screening substances for their ability to interact with UCH-L3 or variant UCH-L3 comprising the steps of providing an UCH-L3 or variant UCH-L3, and testing the ability of selected substances to interact with the UCH-L3 or variant UCH-L3.

In a preferred embodiment, providing an UCH-L3 or variant UCH-L3 is transfecting a host cell with a polynucleotide that encodes an UCH-L3 or variant UCH-L3 to form a transformed cell and maintaining the transformed cell under biological conditions sufficient for expression of the UCH-L3 or variant UCH-L3.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 Sequence alignment of UCH enzymes. Every tenth UCH-L3 residue is delineated with a space. Active site residues (Gln-89, Cys-95, His-169, and Asp-184 of UCH-L3) are indicated by a (*). Other invariant residues are indicated in boxes. Secondary structural elements seen in the UCH-L3 crystal structure are indicated above the sequence, (FIG. 3). Residues that are disordered in the UCH-L3 crystal structure are indicated with broken lines. SwissProt Database entries shown are : UCH-L3, (Human ; SW : P15374) ; UCH-L1, (Human ; SW : P09936) ; UBL- DROME (D. melanogaster ; SW : P35122) ; SCHPO, (S. pombe ; SW : Q10171) ; YUH1, (S. cerevisiae ; SW : P35127).

FIG. 2 Electron Density Map. Electron density map is shown contoured at 1. 0 RMSD with the refined coordinates. Map calculation used B1 (0. 9796 A) structure factor amplitudes in the resolution range 10-2. 35 A. The MAD phases were refined by solvent flattening and histogram shifting. The position of the selenium atom of Met-87 is apparent from the pseudo isomorphous (k3-X1) difference map, which is contoured at 30 RMSD.

FIG. 3 Ribbon diagram of UCH-L3. Side chains of the active site residues, Gln-89, Cys-85, His-169, and Asp-184, are shown by wide vertical lines, and labeled Q, C, H, and D. Amino and Carboxyl termini are denoted with N and C.

Residues 146 and 167, which mark the ends of the large disordered loop, are indicated. Secondary structures were defined by the program PROMOTIF (Hutchinson and Thornton, 1996). Strands are stippled and helices horizontal lines.

Helix 4, which contains the active site nucleophile, Cys-95, is in the center of the diagram. Strand 1 (29-34), strand 2 (49-57), strand 3 (168-176), strand 4 (179-183), strand 5 (191-195), strand 6 (223-229). Helix 1 (residues 13-22), helix 2 (39-42), helix 3 (60-76), helix 4 (92-110), helix 5 (118-125), helix 6 (131-140), helix 7 (201- 215). Helix 4 has two kinks at residues 95 and 105 that separate the large central a-helical segment from the two short 310 segments at the ends of this helix. All other helices are alpha. FIG. 3 and FIG. 5A were made with the programs MOLSCRIPT (Kraulis, 1991) and RASTER 3D (Bacon and Anderson, 1988).

FIG. 4 Comparison of UCH-L3 and Papain-like active sites. A) Active site residues of UCH-L3. Gln-89, Cys-95, His-169, and Asp-184, are shown in thick lines. A representative collection of 8 papain-like enzyme active sites are shown in thin lines following least squares overlap on the active site residue Ca atoms. The papain-like structures shown have PBD identifiers 9pap, 4pad, lpop, 2act, laec, lhuc, Icsb, lgec. Other papain-like structures used in structural comparisons in this paper are : lthe, lcpj, lpad, 2pad, 5pad, 6pad, lstf, lpip, lppp, lpe6, lppd, lppn, Ippo.

Refer to the PBD for primary references to these structures, which are not included here because of space limits.

FIG. 5 Comparison of UCH-L3 with Cathepsin B. A) UCH-L3 (upper) and cathepsin B (lower) shown in a similar orientation as FIG. 3. Equivalent residues were defined by LSQMAN (Kleywegt and Jones, 1994). Pairs of C'atoms were included in the overlap in their separation is less than 3. 0 A and if they form a stretch of at least 5 contiguous residues. Equivalent residues, as defined by LSQMAN, are shown in the horizontal striped ribbon representation, and listed here : residues 32-37 of UCH-L3 : : residues 152-157 of cathepsin B, 48-60 : : 166-178, 84-90 : : 18-24, 92- 106 : : 27-40, 167-174 : : 197-204, 182-186 : : 217-221. B) Topology diagram of secondary structure for (3-sheet and helix 4 of UCH-L3 (upper) and structurally equivalent segments of cathepsin B (lower). Secondary structural elements are marked according to their order of occurrence along the amino acid sequence ; (wide vertical lines, cross-

hatched lines, straight-hatched lines, slanted lines, close vertical lines, wide horizontal lines, close horizontal lines). The main topological difference is for the helix, which in papain-like enzymes is the first of these secondary structural elements in the sequence, while for UCH-L3 helix 4 is found between strands 2 and 3. The long disordered loop of UCH-L3 is indicated with a dotted line.

FIG. 6 Active site clefts of papain-like enzymes and UCH-L3. Orientation is the same as for FIG. 3. A) Glycyl Endopeptidase complex with the inhibitor Benzyloxycarbonyl-L-V-G-Methylene, which occupies the S4, S3, S2 and Sl sites (O'Hara et a/., 1995). B) Cathepsin B with the inhibitor CA030, which occupies the S2, S 1, S 1'and S2'sites (Turk et al, 1995). Protein surfaces are shaded with slanted lines according to curvature. Bound inhibitors are vertical lines. Active site Cys residue is cross-hatched, other active site residues close vertical lines. C) UCH-L3 molecular surface shaded for the invariant residue of FIG. 1. Active site residues are shown in close vertical lines, basic residues close horizontal lines, acidic residue wide vertical lines, polar residues cyan, and hydrophobic residues green. This figure was prepared with the program GRASP (Nicholls et al., 1991).

FIG. 7 Proposed orientation of UCH-L3/Ubiquitin binding. This view is approximately perpendicular (from the left) of FIG. 3. Crystal structure of UCH-L3 is shown with (3-strands slanted lines, helix-4 horizontal lines, and other structure cross- hatched lines. The glycyl endopeptidase and cathepsin B S and S'site inhibitors of FIG. 6A and FIG. 6B are shown in wide vertical lines and close vertical lines respectively after least squares overlap of the papain-like enzyme complexes on the UCH-L3 crystal structure. The structure of ubiquitin (Vijay-Kumar et al., 1987), shown unshaded, has been positioned with the basic face adjacent to UCH-L3, the C-terminal carboxylate adjacent to UCH-L3 Cys-95, and with the flexible C-terminal residues following the path of the Glycyl Endopeptidase S site inhibitor.

FIG. 8 The active site cleft of UCH-L3 is blocked. Stereoview of the UCH-L3 active site in approximately the same orientation as FIG. 3. The active site residues Gln-89, Cys-95, His-169, and Asp-184, are labeled with Q, C, H, and D, respectively. UCH-L3 residues Leu-9, Glu-10, Ala-11, and Ser-92 are labeled.

UCH-L3 is shaded with horizontal lines, with the two segments proposed to move upon binding substrate shaded by slanted lines (residues 9-12 ; 90-94). The S4-S1 site inhibitor of Glycyl Endopeptidase (FIG. 6A) is shown in vertical lines after superposition on the UCH active site residue Ca atoms.

FIG. 9 Possible orientations of the UCH-L3 disordered loop. The crystal structure of UCH-L3 is shown in the same shading representation and orientation as FIG. 7. The docked ubiquitin molecule has been moved slightly away from the UCH-L3 for clarity. Residues that follow ubiquitin in an a-linked substrate adduct have been included in broken horizontal lines. Three possible classes of conformation are shown in close vertical lines, wide horizontal lines, and wide vertical lines, for the disordered loop (residues 147-166) with respect to the substrate.

FIG. 10 Relative rates of hydrolysis of ubiquitin derivatives by UCH isozymes. The rates of hydrolysis were measured by HPLC according to Wilkinson et al. (1986). The brackets [] surround the leaving group. The rates shown are obtained with 15 pM substrates (-20 times Km) and are given as the ratio of rates for the indicated substrate vs. that for ubiquitin ethyl ester. The error bars represent the standard error of the mean (See Table 1 for absolute rates). Note the log scale.

FIG. 11 UbCEP52 is a substrate for UCH-L3. Each lane contains 10 ug of substrate and 1 Rg of enzyme. The time of digestion is given in minutes. A : SDS- PAGE of the reaction time course, protein detected by Coomassie Blue staining. B : Immunoblot of a duplicate gel, probed with rabbit antisera to human CEP52. The unmarked band is a minor contaminant.

FIG. 12 Nucleic acid inhibits the processing of UbCEP52 by UCH-L3.

Nucleic acid was added at a concentration of 0. 05 mg/mL, and incubated for ten minutes with the substrate before enzyme was added to start the reaction. Addition of dsDNA to UbOEt had no effect on the rate of ester hydrolysis (triangles). The rate of hydrolysis of UbCEP52 is only a few-fold slower (+). Addition of RNAse A slightly increased the rate of hydrolysis of UbCEP52 (x). Single stranded DNA had little effect (solid circles), while either E. coli RNA (solid squares), a plasmid DNA (open circles), or a double-stranded 42 bp DNA (open squares) significantly inhibited.

FIG. 13 Co-translational processing of the proubiquitin (left panel) and UbCEP80 (right panel) gene products by UCH-L1 and UCH-L3. The bacterial host BL21 (DE3) was co-transformed with a plasmid encoding the substrates and the Ampr gene product and a second vector encoding the indicated enzyme and Kanr gene product. Protein production was induced with IPTG for three hours and whole cell lysates were subjected to SDS-PAGE and immunoblotting with anti-ubiquitin (left panel) or anti-CEP80 (right panel) antibodies.

FIG. 14 Alignment of Known UCH Sequences The known UCH sequences are aligned in FIG. 14, where only residues found in at least three sequences are indicated by boxes. The numbering system corresponds to the human UCH-L1 residues.

FIG. 15 Ubiquitin Binding By UCH-L3 Shows that purified UCH-L3 is 91% occupied by ubiquitin when chromatographed in the presence of 5 uM ubiquitin-containing buffer. Integration of the peak area shows that 3. 45 nmol of ubiquitin was bound to the 3. 80 nmol of UCH-L3 applied. An apparent binding constant of 0. 5 pM can be calculated from these data.

FIG. 16 Raman Spectra of UCH-L3 from 400 to 1750 cm-1. Two methods were used to calculate the amounts of structural motifs which are based on the

conformationally sensitive nature of the peptide carbonyl stretch absorbance. The spectral bandwidth, intensity, and position of this amide I Stokes emission were used to estimate quantities of four generic secondary structures : helix, (3-sheet, turn, and random (Alix et aL, 1981).

FIG. 17 Temperature-Dependent Changes in the 222 nm CD Signal of UCH-L1 A) A thermal transition at approximately 52°C results in a 45% diminishment in this conformationally sensitive signal. UCH-L3 is also subject to the same transition, though the loss of ellipticity is slightly less. Cooling the sample results in the restoration of the original spectra, and wavelength scans at 65°C are typical of proteins with high random coil content. B) Shows the Arrhenius plot of the data. As obtained from the replot, this transition is characterized by values of AH= 1. 56 kJ/mol of residue, AS = 4. 80 J/K mol of residue, and AG = 28. 6 kJ/mol of UCH-L1 at 25°C.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS The present inventors have determined the UCH-L3 crystal structure in atomic detail, which provides the basis for altering the active site of the protein. UCH-L3 belongs to a family of UCH (ubiquitin C-terminal hydrolase) enzymes that all catalyze the removal of adducts from the C-terminus of the small protein ubiquitin. Because the similarity in amino acid sequences indicates that all of the UCH enzymes will have the same three dimensional structure, and because UCH-L3 is the first UCH for which a structure is known, the present invention is applicable to all UCH enzymes.

-UCH-L3 has a core catalytic structure that strongly resembles cathepsin B, a papain-like protease. The active site groove is occluded by two loops, and it is postulated that a substrate-induced conformational change is required to clear the cleft

and allow access to the active-site cysteine. Thus, only ubiquitin derivatives are substrates because only they can form the extensive interactions with the S'site required to trigger the necessary conformational change generating the active conformation of the enzyme.

Specificity for P'residues must be determined by the residues lining the corresponding S'sites on the UCH enzymes. The sequence of these proteins varies widely in several areas, including a region just N-terminal to the active site histidine.

This sequence is disordered in the UCH-L3 structure, but may be positioned to form a significant contact region with the P'residues of substrates. Thus, it is likely that this hypervariable region is important in determining substrate selectivity and the somewhat shorter loop near the active site cysteine in UCH-L1 restricts the possible substrates by conferring a narrower or more restricted active site cleft.

UCH enzymes have potential in the commercial production of peptides (and possibly proteins), that are initially expressed as ubiquitin fusions from which the ubiquitin is later cleaved by UCH activity. UCH-L3 is already used in this way for the production of peptides. See, for example, U. S. Patent No. 5, 620, 923. The utility of this process could be enhanced by the availability of a UCH enzyme that possess greater thermal and/or chemical stability.

Knowledge of the UCH-L3 structure can be used to design variants with enhanced properties such as increased stability. This work can be performed by inspection of the UCH-L3 structure on a graphics workstation, by computer manipulation of UCH-L3 coordinates, and calculations such as energy minimization.

Variants whose potential properties are initially predicted in light of the UCH-L3 structure can be produced by the usual techniques of molecular biology.

Enhanced stability might result from increasing the number of salt bridge or hydrogen bonding interactions, improving the packing of hydrophobic groups, or by

adding disulfide bonds. Chemical stability might be improved by replacement of chemically labile groups with more stable amino acid residues. For example, buried methionine residues might be replaced with the more inert leucine amino acid residue.

Cysteine residues might also be replaced, for example with alanine or serine side chains.

The present invention provides teaching to replace an active site residue, such as cysteine-95, with another amino acid residue to produce a more stable enzyme that uses a serine protease mechanism rather than the cysteine protease mechanism of wild type UCH enzymes. For example, the present invention provides guidance to make other changes in the enzyme structure, such changing aspartic acid to asparagine, to alter the specificity or stability of the enzyme. All such approaches to produce a more stable enzyme will be assisted by knowledge of the UCH-L3 structure.

Because ubiquitin chemistry is of fundamental importance to cellular metabolism, it may be possible to design therapeutic agents that function through modification of UCH activities. For example, a specific UCH inhibitor may increase (or reduce) the rate at which a protein (s) is degraded by the 26S protease. Because some proteins that function in proliferation are normally turned over by ubiquitin- mediated degradation, UCH inhibitors may have utility in the treatment of cancers.

Another possible utility is in the treatment of wasting diseases which are thought to result from excessive ubiquitin-mediated proteolysis. UCH inhibitors may also find utility in the treatment of neurodegenerative diseases, since the UCH-L1 isozyme is highly abundant in neuronal tissue, and these diseases are characterized by deposits that are rich in ubiquitin conjugates (i. e. UCH substrates).

Ubiquitin C-terminal hydrolases catalyze the removal of adducts from the C-terminus of ubiquitin. The present inventors have determined the crystal structure of the recombinant human ubiquitin C-terminal hydrolase, UCH-L3, by X-ray crystallography at 1. 8 A resolution. The structure is comprised of a central

antiparallel-sheet flanked on both sides by a-helices. The P-sheet and one of the helices resemble the well known papain-like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH-L3 active site catalytic triad of Cys-95, His-169 and Asp-184, and the oxyanion hole residue Gln-89. Papain and UCH-L3 differ, however, in strand and helix connectivity, which in the UCH-L3 structure includes a disordered 20-residue loop (res 147-166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain-like enzymes, the inventors set forth the following mechanism to describe the binding of ubiquitin to UCH-L3. The UCH- L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (res 9-12 and 90-94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non-specific hydrolysis.

Crystallization The recombinant human UCH-L3 used in these studies was purified as described (Larsen et a/., 1996). The protein solution used in crystallization trials was 12 mg/mL UCH-L3 in 50 mM Tris Hcl, pH 7. 6, 15 mM BME, 1 mM EDTA. This solution was stored in aliquots at-70°C. Crystallization was performed at 4°C in sitting drops. The reservoir solution was 26% (w/w) PEG 4000, 200 mM sodium acetate, 100 mM Pipes pH 6. 7, and 10 mM DTT. The drop solution was 3 uL of protein solution mixed with 3 uL of reservoir solution. These conditions produced crystalline aggregates after 4-5 days.

Single crystals were obtained by microseeding. One of the initial aggregates was ground up with a needle, and the needle streaked through a drop that was identical to the conditions described above, but which had equilibrated for 3-5 days.

Small single crystals appeared after several days.

Large crystals were obtained by macroseeding. Using a rayon loop, a small single crystal was transferred into reservoir solution, allowed to wash for several

minutes, and then transferred into another drop that has been equilibrated for 3-5 days.

The same reservoir and drop condition used to obtain the initial aggregates were also used for the subsequent micro and macroseeding. The crystals attain their maximum size in 5-10 days following macroseeding. Typical crystal dimensions are 0. 3 mm x 0. 3 mm x 0. 6 mm.

For generation of selenomethionine-substituted UCH-L3 (SeUCH-L3), the gal-, met- auxotroph B834 (DE3) of the BL21 strain (Studier and Moffatt, 1986) harboring pRSL3 (Larsen et al., 1996) was grown on LB agar as colonies. A single colony was inoculated into 50 mL LB media and grown overnight, followed by dilution into 6 liters of modified M9 media. Solutions O, P, S, and V (Weber et al., 1992), uracil (Final concentration of 1 mM), and selenomethionine (final concentration of 50 pg/l) were sterile filtered and added to M9 media.

At an OD6oonm of 0. 6, the cells were induced with 0. 5 mM IPTG for three hours before harvesting by centrifugation. Purification of SeUCH-L3 was the same as for wild type. Ion electrospray mass spectrometry showed an incorporation of >98% Se at each Met codon. SeUCH-L3 and wild type UCH-L3 have comparable specific activities. SeUCH-L3 crystals were grown under the same conditions as native protein, although in this case the seeding steps proved unnecessary and growth time from initial set up was 5-10 days.

Data Collection and Processing The native and SeUCH-L3 crystals are isomorphous ; space group P212121, cell dimensions : a=48. 6 A, b=60. 8 A, c=81. 4 A. There is one molecule in the asymmetric unit, and the Matthew's parameter, Vm, is 2. 37 Å3Da, which corresponds to a solvent content of 48% (Matthews, 1968).

All data were collected at 100K. Prior to cryocooling the crystals were transferred to the reservoir solution, and then to a series of solutions that were

identical except for 2% increments in glycerol concentration up to a final concentration of 18% glycerol. The cryoprotected crystals were suspended in a rayon loop and cooled by plunging into liquid nitrogen.

Multiwavelength data were collected from a single SeUCH-L3 crystal on a MAR imaging plate detector at beamline X12C of the National Synchrotron Light Source, Brookhaven National Laboratory. The three wavelengths collected were selected from the fluorescence spectrum ; % l (0. 9796 A) was chosen as the inflection, or rise, corresponding to the minimum value of f'; B2 (0. 9793 A) was taken as the peak, corresponding to the maximum in f"; B3 (0. 9300 A) was chosen for the remote wavelength, corresponding to the maximum in f. Data from each wavelength were indexed and integrated independently, and data from all three wavelengths were scaled together from 6. 0 A to 2. 2 A. The resulting scale factors were then applied separately to each individual wavelength for data from 30 A to 2. 35 A. Data from a native crystal were collected to 1. 8 A resolution on a MAR imaging plate detector at beamline 7-1 of the Stanford Synchrotron Radiation Laboratory, Palo Alto. All data were processed with the programs DENZO and SCALEPAK (Otwinowski, 1993).

See Table 1 for data statistics.

Table 1. Data Processing Statistics Native . 1 2 ? 3 SSRL NSLSa NSLS NSLS Wavelength (A) 1. 080 0. 9796 0. 9793 0. 930 Resolution limit (A) 1. 80 2. 35 2. 35 2. 35 High resol. shell (A) (1. 83-1. 80) (2. 43-2. 35) (2. 43- (2. 43-2. 35) 2. 35) #Unique reflections 23334 11282 11217 11108 Completeness (%) 98 (93) 96 (86) 90 (84) 89 (91) <I/#(I)> 20 (5) 15 (5) 15 (5) 15 (6) Redundancy'4. 5 (3) 2 (1) 2 (1) 2 (1. 5)

R sym (%) c 4. 0 (19) 4. 9 (13. 2) 4. 4 (12. 1) 4. 8 (13. 7) Mosaicity (°) 1. 18 0. 42 0. 42 0. 42 a Data were collected on MAR imaging plate detectors on beamline 7-1 at the Stanford Synchrotron Radiation Laboratory (SSRL) or beamline X12C of the National Synchrotron Light Source (NSLS).

Redundancy is defined as the ratio of observed/unique structure factor amplitudes. c R sym = 100 * EhkuiIl ; _<I> = E<I> Data were processed with DENZO and SCALEPACK (Otwinowski, 1993).

Structure Determination and Refinement Crystallographic computing was performed using programs from the CCP4 suite (CCP4, 1994), unless otherwise stated. Of the seven methionine residues in UCH-L3, all except the amino terminal Met are ordered. The six selenium sites were identified from difference Patterson and Fourier functions using the program XtalView (McRee, 1992). Selenium parameters were refined in MLPHARE (Otwinowski, 1991), treating Xl as the native data of a conventional multiple isomorphous phase determination (Ramakrishnan and Biou, 1997). The mean figure of merit calculated by MLPHARE was 0. 42.

Phases computed with MLPHARE were refined by solvent flattening and histogram shifting with the program DM (Cowtan, 1994) to a mean figure of merit of 0. 77. The resulting electron density map was readily interpretable for the majority of the UCH-L3 sequence, see FIG. 2. Rounds of refinement with XPLOR (Brunger, 1992b) were interspersed with mode building (Jones et al., 1991). XI amplitudes from 10. 0 A to 2. 35 A resolution were used in the refinement, with phase restraints also applied. At this stage the Rvalue against 10. 0 A to 2. 35 A data was 24. 3% and the free Rvalue was 30. 4% (Brunger, 1992a). No sigma cuts were applied to refinement or Rvalue calculations.

Refinement was continued against 6. 0 A to 1. 8 A data collected from a native crystal (see Table 2). Because of a slight deviation from true isomorphism between

the native and SeUCH-L3 crystals, phase restrains were not employed for the high resolution refinement. The final model includes 121 water molecules and 205 of the total 230 UCH-L3 residues. The current Rvalue is 23. 0% and the free Rvalue is 28. 6%. The first four residues at the amino-terminus are disordered, as are residues 147-166 and 218. The model has good stereochemistry as judged by PROCHECK (Laskowski et al., 1993).

UCH-L3 COORDINATES REMARK FILENAME="134_reb_8_bref. pdb" REMARK TOPHI 9. pep-MACRO for protein sequence REMARK DATE : 14-Mar-97 22 : 45 : 32 created by user : stemmler ATOM 1 CB ARG 5 34. 943 15. 749 56. 409 1. 00 29. 03 AAAA ATOM 2 CG ARG 5 34. 137 16. 647 57. 309 1. 00 30. 95 AAAA ATOM 3 CD ARG 5 34. 789 16. 934 58. 656 1. 00 32. 26 AAAA ATOM 4 NE ARG 5 34. 211 18. 164 59. 198 1. 00 36. 00 AAAA ATOM 5 HE ARG 5 33. 836 18. 803 58. 557 1. 00 0. 00 AAAA ATOM 6 CZ ARG 5 34. 155 18. 493 60. 486 1. 00 36. 77 AAAA ATOM 7 NH1 ARG 5 33. 606 19. 646 60. 843 1. 00 38. 66 AAAA ATOM 8 HH11 ARG 5 33. 219 20. 248 60. 144 1. 00 0. 00 AAAA ATOM 9 HH12ARG 5 33. 563 19. 905 61. 809 1. 00 0. 00 AAAA ATOM 10 NH2 ARG 5 34. 687 17. 707 61. 408 1. 00 36. 16 AAAA ATOM 11 HH21 ARG 5 35. 156 16. 864 61. 142 1. 00 0. 00 AAAA ATOM 12 HH22ARG 5 34. 619 17. 958 62. 372 1. 00 0. 00 AAAA ATOM 13 C ARG 5 34. 702 17. 197 54. 397 1. 00 26. 94 AAAA ATOM 14 0 ARG 5 33. 828 18. 051 54. 424 1. 00 24. 12 AAAA ATOM 15 HT1 ARG 5 36. 170 14. 703 54. 248 1. 00 0. 00 AAAA ATOM 16 HT2 ARG 5 34. 87914. 767 53. 128 1. 00 0. 00 AAAA ATOM 17 N ARG 5 35. 131 14. 709 54. 140 1. 00 34. 97 AAAA ATOM 18 HT3 ARG 5 34. 83813. 776 54. 488 1. 00 0. 00 AAAA ATOM 19 CA ARG 5 34. 460 15. 792 54. 952 1. 00 30. 10 AAAA ATOM 20 N TRP 6 35. 923 17. 431 53. 940 1. 00 25. 41 AAAA ATOM 21 H TRP 6 36. 59516. 737 53. 823 1. 00 0. 00 AAAA ATOM 22 CA TRP 6 36. 37418. 767 53. 614 1. 00 22. 76 AAAA ATOM 23 CB TRP 6 37. 836 18. 901 53. 993 1. 00 20. 39 AAAA ATOM 24 CG TRP 6 38. 11718. 460 55. 390 1. 00 22. 77 AAAA ATOM 25 CD2 TRP 6 37. 623 19. 059 56. 594 1. 00 20. 61 AAAA

ATOM 26 CE2 TRP 6 38. 108 18. 291 57. 671 1. 00 21. 75 AAAA ATOM 27 CE3 TRP 6 36. 823 20. 177 56. 860 1. 00 18. 56 AAAA ATOM 28 CD1 TRP 6 38. 875 17. 392 55. 778 1. 00 23. 73 AAAA ATOM 29 NE1 TRP 6 38. 87217. 283 57. 144 1. 00 23. 09 AAAA ATOM 30 HE1 TRP 6 39. 332 16. 602 57. 685 1. 00 0. 00 AAAA ATOM 31 CZ2 TRP 6 37. 816 18. 594 59. 002 1. 00 20. 20 AAAA ATOM 32 CZ3 TRP 6 36. 537 20. 483 58. 184 1. 00 21. 88 AAAA ATOM 33 CH2 TRP 6 37. 032 19. 690 59. 238 1. 00 20. 85 AAAA ATOM 34 C TRP 6 36. 187 19. 085 52. 141 1. 00 22. 14 AAAA ATOM 35 0 TRP 6 36. 015 18. 194 51. 321 1. 00 24. 44 AAAA ATOM 36 N LEU 7 36. 200 20. 366 51. 815 1. 00 20. 82 AAAA ATOM 37 H LEU 7 36. 125 21. 021 52. 539 1. 00 0. 00 AAAA ATOM 38 CA LEU 7 36. 317 20. 802 50. 423 1. 00 24. 47 AAAA ATOM 39 CB LEU 7 36.404 22.327 50.367 1.00 24. 34 AAAA ATOM 40 CG LEU 7 36.146 23.017 49.040 1.00 22. 59 AAAA ATOM 41 CD1 LEU 7 34.687 22.829 48.654 1.00 26. 54 AAAA ATOM 42 CD2 LEU 7 36. 497 24. 483 49. 174 1. 00 21. 09 AAAA ATOM 43 C LEU 7 37.567 20.197 49.771 1.00 23. 51 AAAA ATOM 44 0 LEU 7 38. 653 20. 247 50. 341 1. 00 25. 30 AAAA ATOM 45 N PRO 8 37.403 19.505 48.638 1.00 26. 01 AAAA ATOM 46 CD PRO 8 36. 137 19. 093 48. 006 1. 00 27. 47 AAAA ATOM 47 CA PRO 8 38. 559 18. 953 47. 929 1. 00 25. 82 AAAA ATOM 48 CB PRO 8 37. 93518. 297 46. 701 1. 00 27. 75 AAAA ATOM 49 CG PRO 8 36. 553 17. 925 47. 172 1. 00 27. 83 AAAA ATOM 50 C PRO 8 39. 570 20. 034 47. 550 1. 00 24. 44 AAAA ATOM 51 0 PRO 8 39. 217 21. 195 47. 386 1. 00 22. 60 AAAA ATOM 52 N LEU 9 40. 829 19. 647 47. 415 1. 00 27. 15 AAAA ATOM 53 H LEU 9 41. 016 18. 683 47. 463 1. 00 0. 00 AAAA ATOM 54 CA LEU 9 41. 900 20. 614 47. 200 1. 00 26. 84 AAAA ATOM 55 CB LEU 9 43.179 20.15 47.910 1.00 26. 91 AAAA ATOM 56 CG LEU 9 42. 992 19. 872 49. 410 1. 00 26. 00 AAAA ATOM 57 CD1 LEU 9 44. 33719. 580 50. 023 1. 00 27. 63 AAAA ATOM 58 CD2 LEU 9 42. 319 21. 062 50. 114 1. 00 21. 92 AAAA ATOM 59 C LEU 9 42.145 20.885 45.712 1.00 26. 78 AAAA ATOM 60 O LEU 9 43.133 20.437 45.119 1.00 26. 81 AAAA ATOM 61 N GLU 10 41. 168 21. 542 45. 102 1. 00 26. 31 AAAA ATOM 62 H GLU 10 40. 351 21. 729 45. 616 1. 00 0. 00 AAAA ATOM 63 CA GLU 10 41.244 21.971 43.716 1.00 27. 90 AAAA ATOM 64 CB GLU 10 40. 755 20. 874 42. 772 1. 00 33. 87 AAAA ATOM 65 CG GLU 10 39. 629 20. 026 43. 317 1. 00 45. 72 AAAA ATOM 66 CD GLU 10 39. 141 18. 967 42.323 1.00 53. 31 AAAA

ATOM 67 OE1 GLU 10 38. 277 19. 304 41. 469 1. 00 52. 71 AAAA ATOM 68 OE2 GLU 10 39. 590 17. 796 42. 437 1. 00 55. 78 AAAA ATOM 69 C GLU 10 40. 369 23. 186 43. 571 1. 00 25. 52 AAAA ATOM 70 0 GLU 10 39. 368 23. 305 44. 268 1. 00 25. 82 AAAA ATOM 71 N ALA 11 40. 835 24. 158 42. 798 1. 00 27. 87 AAAA ATOM 72 H ALA 11 41. 697 23. 992 42. 356 1. 00 0. 00 AAAA ATOM 73 CA ALA 11 40. 094 25. 395 42. 580 1. 00 26. 92 AAAA ATOM 74 CB ALA 11 41. 001 26. 443 42. 011 1. 00 26. 26 AAAA ATOM 75 C ALA 11 38. 935 25. 121 41. 620 1. 00 30. 85 AAAA ATOM 76 0 ALA 11 39. 117 25. 060 40. 401 1. 00 35. 58 AAAA ATOM 77 N ASN 12 37. 764 24. 878 42. 193 1. 00 29. 56 AAAA ATOM 78 H ASN 12 37. 726 24. 985 43. 160 1. 00 0. 00 AAAA ATOM 79 CA ASN 12 36. 591 24. 423 41. 462 1. 00 28. 53 AAAA ATOM 80 CB ASN 12 36. 368 22. 936 41. 738 1. 00 29. 60 AAAA ATOM 81 CG ASN 12 35. 298 22. 334 40. 870 1. 00 30. 93 AAAA ATOM 82 OD1 ASN 12 34. 252 22. 940 40. 629 1. 00 33. 85 AAAA ATOM 83 ND2 ASN 12 35. 509 21. 098 40. 467 1. 00 34. 38 AAAA ATOM 84 HD21 ASN 12 36. 288 20. 611 40. 798 1. 00 0. 00 AAAA ATOM 85 HD22ASN 12 34. 848 20. 762 39. 828 1. 00 0. 00 AAAA ATOM 86 C ASN 12 35. 358 25. 225 41. 891 1. 00 24. 71 AAAA ATOM 87 0 ASN 12 34. 693 24. 887 42. 867 1. 00 25. 47 AAAA ATOM 88 N PRO 13 35. 065 26. 321 41. 181 1. 00 23. 24 AAAA ATOM 89 CD PRO 13 35. 778 26. 765 39. 971 1. 00 22. 18 AAAA ATOM 90 CA PRO 13 33. 963 27. 223 41. 515 1. 00 23. 35 AAAA ATOM 91 CB PRO 13 33. 883 28. 143 40. 303 1. 00 24. 69 AAAA ATOM 92 CG PRO 13 35. 244 28. 138 39. 750 1. 00 23. 36 AAAA ATOM 93 C PRO 13 32. 633 26. 507 41. 770 1. 00 23. 21 AAAA ATOM 94 0 PRO 13 31. 939 26. 820 42. 720 1. 00 23. 70 AAAA ATOM 95 N GLU 14 32. 310 25. 502 40. 966 1. 00 26. 47 AAAA ATOM 96 H GLU 14 32. 975 25. 148 40. 341 1. 00 0. 00 AAAA ATOM 97 CA GLU 14 30. 975 24. 917 41. 027 1. 00 31. 39 AAAA ATOM 98 CB GLU 14 30. 666 24. 117 39. 757 1. 00 38. 34 AAAA ATOM 99 CG GLU 14 29. 181 23. 754 39. 611 1. 00 51. 49 AAAA ATOM 100 CD GLU 14 28. 796 23. 371 38. 181 1. 00 60. 13 AAAA ATOM 101 OE1 GLU 14 28. 176 24. 206 37. 473 1. 00 62. 58 AAAA ATOM 102 OE2 GLU 14 29. 120 22. 230 37. 766 1. 00 63. 49 AAAA ATOM 103 C GLU 14 30. 751 24. 051 42. 267 1. 00 28. 36 AAAA ATOM 104 0 GLU 14 29. 693 24. 131 42. 895 1. 00 26. 17 AAAA ATOM 105 N VAL 15 31. 749 23. 241 42. 618 1. 00 25. 73 AAAA ATOM 106 H VAL 15 32. 517 23. 168 42. 008 1. 00 0. 00 AAAA ATOM 107 CA VAL 15 31. 733 22. 474 43. 869 1. 00 24. 08 AAAA

ATOM 108 CB VAL 15 32. 95621. 511 43. 945 1. 00 23. 74 AAAA ATOM 109 CG1 VAL 15 33. 007 20. 829 45. 290 1. 00 25. 22 AAAA ATOM 110 CG2 VAL 15 32. 860 20. 440 42. 849 1. 00 19. 71 AAAA ATOM 111 C VAL 15 31. 712 23. 397 45. 109 1. 00 22. 96 AAAA ATOM 112 0 VAL 15 30. 904 23. 217 46. 023 1. 00 22. 19 AAAA ATOM 113 N THR 16 32. 537 24. 440 45. 089 1. 00 22. 52 AAAA ATOM 114 H THR 16 33. 20124. 524 44. 368 1. 00 0. 00 AAAA ATOM 115 CA THR 16 32. 530 25. 436 46. 156 1. 00 20. 27 AAAA ATOM 116 CB THR 16 33. 633 26. 458 45. 926 1. 00 18. 24 AAAA ATOM 117 OG1 THR 16 34. 835 25. 756 45. 605 1. 00 18. 68 AAAA ATOM 118 HG1 THR 16 35. 579 26. 372 45. 533 1. 00 0. 00 AAAA ATOM 119 CG2 THR 16 33. 858 27. 292 47. 169 1. 00 17. 57 AAAA ATOM 120 C THR 16 31. 192 26. 164 46. 315 1. 00 22. 22 AAAA ATOM 121 0 THR 16 30. 700 26. 352 47. 440 1. 00 19. 76 AAAA ATOM 122 N ASN 17 30. 589 26. 550 45. 192 1. 00 22. 44 AAAA ATOM 123 H ASN 17 31. 067 26. 433 44. 350 1. 00 0. 00 AAAA ATOM 124 CA ASN 17 29. 268 27. 190 45. 197 1. 00 22. 07 AAAA ATOM 125 CB ASN 17 28. 932 27. 721 43. 807 1. 00 22. 34 AAAA ATOM 126 CG ASN 17 29. 788 28. 905 43. 424 1. 00 23. 06 AAAA ATOM 127 OD1 ASN 17 30. 205 29. 679 44. 282 1. 00 21. 72 AAAA ATOM 128 ND2 ASN 17 30.007 29.089 42.129 1.00 20. 19 AAAA ATOM 129 HD21 ASN 17 29. 565 28. 488 41. 489 1. 00 0. 00 AAAA ATOM 130 HD22ASN 17 30. 606 29. 826 41. 897 1. 00 0. 00 AAAA ATOM 131 C ASN 17 28. 165 26. 234 45. 669 1. 00 20. 44 AAAA ATOM 132 0 ASN 17 27. 300 26. 625 46. 449 1. 00 20. 46 AAAA ATOM 133 N GLN 18 28. 276 24. 961 45. 316 1. 00 20. 08 AAAA ATOM 134 H GLN 18 28. 974 24. 713 44. 675 1. 00 0. 00 AAAA ATOM 135 CA GLN 18 27. 358 23. 958 45. 853 1. 00 24. 90 AAAA ATOM 136 CB GLN 18 27. 549 22. 612 45. 148 1. 00 30. 06 AAAA ATOM 137 CG GLN 18 26. 740 22. 490 43. 862 1. 00 42. 05 AAAA ATOM 138 CD GLN 18 25. 343 23. 105 43. 988 1. 00 51. 77 AAAA ATOM 139 OE1 GLN 18 24. 448 22. 521 44. 607 1. 00 55. 04 AAAA ATOM 140 NE2 GLN 18 25. 173 24. 317 43. 456 1. 00 52. 02 AAAA ATOM 141 HE21 GLN 18 25. 902 24. 740 42. 952 1. 00 0. 00 AAAA ATOM 142 HE22 GLN 18 24. 293 24. 702 43. 641 1. 00 0. 00 AAAA ATOM 143 C GLN 18 27. 471 23. 778 47. 367 1. 00 26. 45 AAAA ATOM 144 0 GLN 18 26. 453 23. 712 48. 068 1. 00 21. 63 AAAA ATOM 145 N PHE 19 28. 705 23. 783 47. 875 1. 00 26. 95 AAAA ATOM 146 H PHE 19 29. 452 23. 787 47. 242 1. 00 0. 00 AAAA ATOM 147 CA PHE 19 28. 967 23. 707 49. 317 1. 00 24. 56 AAAA ATOM 148 CB PHE 19 30. 471 23. 527 49. 544 l. OO 28. 12 AAAA

ATOM 149 CG PHE 19 30. 836 23. 135 50. 950 1. 00 30. 72 AAAA ATOM 150 CD1 PHE 19 30. 010 22. 278 51. 692 1. 00 30. 11 AAAA ATOM 151 CD2 PHE 19 32. 035 23. 577 51. 516 1. 00 29. 96 AAAA ATOM 152 CE1 PHE 19 30. 383 21. 862 52. 980 1. 00 31. 40 AAAA ATOM 153 CE2 PHE 19 32. 415 23. 167 52. 805 1. 00 29. 30 AAAA ATOM 154 CZ PHE 19 31. 591 22. 307 53. 533 1. 00 28. 78 AAAA ATOM 155 C PHE 19 28. 450 24. 948 50. 088 1. 00 22. 86 AAAA ATOM 156 0 PHE 19 27. 789 24. 822 51. 121 1. 00 20. 58 AAAA ATOM 157 N LEU 20 28. 672 26. 130 49. 525 1. 00 19. 47 AAAA ATOM 158 H LEU 20 29. 27126. 150 48. 752 1. 00 0. 00 AAAA ATOM 159 CA LEU 20 28. 088 27. 368 50. 037 1. 00 19. 77 AAAA ATOM 160 CB LEU 20 28. 421 28. 518 49. 088 1. 00 18. 13 AAAA ATOM 161 CG LEU 20 29. 828 29. 107 49. 129 1. 00 19. 84 AAAA ATOM 162 CD1 LEU 20 29. 857 30. 462 48. 468 1. 00 15. 91 AAAA ATOM 163 CD2 LEU 20 30. 234 29. 246 50. 567 1. 00 23. 06 AAAA ATOM 164 C LEU 20 26. 560 27. 271 50. 197 1. 00 22. 72 AAAA ATOM 165 0 LEU 20 25. 986 27. 680 51. 211 1. 00 21. 79 AAAA ATOM 166 N LYS 21 25. 907 26. 748 49. 171 1. 00 22. 10 AAAA ATOM 167 H LYS 21 26. 42126. 490 48. 376 1. 00 0. 00 AAAA ATOM 168 CA LYS 21 24. 474 26. 520 49. 220 1. 00 23. 06 AAAA ATOM 169 CB LYS 21 24. 026 25. 887 47. 900 1. 00 25. 93 AAAA ATOM 170 CG LYS 21 22. 528 25. 662 47. 768 1. 00 35. 03 AAAA ATOM 171 CD LYS 21 22. 270 24. 673 46. 651 1. 00 43. 04 AAAA ATOM 172 CE LYS 21 20. 819 24. 706 46. 198 1. 00 49. 98 AAAA ATOM 173 NZ LYS 21 20. 563 23. 681 45. 137 1. 00 53. 62 AAAA ATOM 174 HZ1 LYS 21 20. 728 22. 728 45. 519 1. 00 0. 00 AAAA ATOM 175 HZ2 LYS 21 21. 199 23. 859 44. 333 1. 00 0. 00 AAAA ATOM 176 HZ3 LYS 21 19. 576 23. 773 44. 822 1. 00 0. 00 AAAA ATOM 177 C LYS 21 24. 124 25. 607 50. 397 1. 00 19. 59 AAAA ATOM 178 0 LYS 21 23. 266 25. 906 51. 220 1. 00 24. 24 AAAA ATOM 179 N GLN 22 24. 836 24. 508 50. 499 1. 00 20. 54 AAAA ATOM 180 H GLN 22 25. 555 24. 352 49. 846 1. 00 0. 00 AAAA ATOM 181 CA GLN 22 24. 549 23. 510 51. 504 1. 00 22. 64 AAAA ATOM 182 CB GLN 22 25. 314 22. 252 51. 121 1. 00 24. 37 AAAA ATOM 183 CG GLN 22 25. 554 21. 226 52. 180 1. 00 35. 79 AAAA ATOM 184 CD GLN 22 26. 685 20. 299 51. 773 1. 00 42. 90 AAAA ATOM 185 OE1 GLN 22 27. 008 20. 178 50. 584 1. 00 45. 37 AAAA ATOM 186 NE2 GLN 22 27. 353 19. 719 52. 751 1. 00 44. 56 AAAA ATOM 187 HE21 GLN 22 27. 195 19. 868 53. 698 1. 00 0. 00 AAAA ATOM 188 HE22 GLN 22 28. 038 19. 097 52. 405 1. 00 0. 00 AAAA ATOM 189 C GLN 22 24. 873 24. 011 52. 924 1. 00 25. 82 AAAA

ATOM 190 O GLN 22 24. 161 23. 680 53. 878 1. 00 25. 10 AAAA ATOM 191 N LEU 23 25. 756 25. 004 53. 011 1. 00 24. 28 AAAA ATOM 192 H LEU 23 26. 233 25. 265 52. 199 1. 00 0. 00 AAAA ATOM 193 CA LEU 23 26. 068 25. 649 54. 289 1. 00 22. 60 AAAA ATOM 194 CB LEU 23 27. 476 26. 249 54. 240 1. 00 23. 15 AAAA ATOM 195 CG LEU 23 28. 621 25. 216 54. 126 1. 00 27. 13 AAAA ATOM 196 CD1 LEU 23 29. 966 25. 935 53. 935 1. 00 22. 36 AAAA ATOM 197 CD2 LEU 23 28. 650 24. 292 55. 360 1. 00 22. 91 AAAA ATOM 198 C LEU 23 25. 046 26. 722 54. 690 1. 00 21. 30 AAAA ATOM 199 0 LEU 23 25. 134 27. 307 55. 770 1. 00 19. 91 AAAA ATOM 200 N GLY 24 24. 102 27. 009 53. 792 1. 00 22. 47 AAAA ATOM 201 H GLY 24 24. 162 26. 600 52. 902 1. 00 0. 00 AAAA ATOM 202 CA GLY 24 22. 979 27. 880 54. 123 1. 00 18. 64 AAAA ATOM 203 C GLY 24 23. 062 29. 291 53. 583 1. 00 20. 19 AAAA ATOM 204 0 GLY 24 22. 359 30. 187 54. 058 1. 00 21. 11 AAAA ATOM 205 N LEU 25 23. 917 29. 497 52. 582 1. 00 21. 94 AAAA ATOM 206 N LEU 25 24. 454 28. 744 52. 264 1. 00 0. 00 AAAA ATOM 207 CA LEU 25 24. 070 30. 802 51. 936 1. 00 22. 38 AAAA ATOM 208 CB LEU 25 25. 525 31. 053 51. 514 1. 00 21. 52 AAAA ATOM 209 CG LEU 25 26. 611 31. 599 52. 432 1. 00 24. 11 AAAA ATOM 210 CD1 LEU 25 26. 183 32. 924 52. 976 1. 00 26. 31 AAAA ATOM 211 CD2 LEU 25 26. 911 30. 589 53. 536 1. 00 25. 31 AAAA ATOM 212 C LEU 25 23. 218 30. 870 50. 670 1. 00 23. 83 AAAA ATOM 213 0 LEU 25 23. 382 30. 069 49. 744 1. 00 22. 04 AAAA ATOM 214 N HIS 26 22. 437 31. 935 50. 568 1. 00 27. 33 AAAA ATOM 215 H HIS 26 22. 373 32. 444 51. 402 1. 00 0. 00 AAAA ATOM 216 CA HIS 26 21. 827 32. 302 49. 297 1. 00 30. 72 AAAA ATOM 217 CB HIS 26 20. 858 33. 461 49. 519 1. 00 35. 03 AAAA ATOM 218 CG HIS 26 19. 725 33. 126 50. 440 1. 00 39. 71 AAAA ATOM 219 CD2 HIS 26 19. 605 33. 253 51. 781 1. 00 38. 99 AAAA ATOM 220 ND1 HIS 26 18.546 32.562 49.999 1.00 40. 72 AAAA ATOM 221 HD1 HIS 26 18. 332 32. 241 49. 089 1. 00 0. 00 AAAA ATOM 222 CE1 HIS 26 17. 743 32. 369 51. 032 1. 00 39. 92 AAAA ATOM 223 NE2 HIS 26 18.359 32.785 52.123 1.00 39. 81 AAAA ATOM 224 HE2 HIS 26 18. 134 32. 577 53. 054 1. 00 0. 00 AAAA ATOM 225 C HIS 26 22. 910 32. 705 48. 286 1. 00 30. 28 AAAA ATOM 226 0 HIS 26 23. 89933. 337 48. 655 1. 00 32. 24 AAAA ATOM 227 N PRO 27 22. 681 32. 456 46. 989 1. 00 28. 45 AAAA ATOM 228 CD PRO 27 21. 500 31. 803 46. 395 1. 00 30. 37 AAAA ATOM 229 CA PRO 27 23. 753 32. 629 46. 003 1. 00 24. 28 AAAA ATOM 230 CB PRO 27 23.367 31.657 44.904 1.00 27. 20 AAAA

ATOM 231 CG PRO 27 21. 850 31. 731 44. 917 1. 00 31. 25 AAAA ATOM 232C PRO2723. 85634. 04445. 4651. 0023. 59 AAAA ATOM 233 0 PRO 27 23. 941 34. 235 44. 267 1. 00 28. 42 AAAA ATOM 234 N ASN 28 23. 971 35. 021 46. 349 1. 00 21. 98 AAAA ATOM 235 H ASN 28 24. 029 34. 719 47. 277 1. 00 0. 00 AAAA ATOM 236 CA ASN 28 24.197 36.393 45.911 1.00 25. 40 AAAA ATOM 237 CB ASN 28 23. 630 37. 402 46. 913 1. 00 29. 65 AAAA ATOM 238 CG ASN 28 23. 848 36. 989 48. 341 1. 00 36. 22 AAAA ATOM 239 OD1 ASN 28 24. 378 37. 743 49. 162 1. 00 39. 03 AAAA ATOM 240 ND2 ASN 28 23. 289 35. 853 48. 689 1. 00 39. 95 AAAA ATOM 241 HD21 ASN 28 23. 560 35. 562 49. 587 1. 00 0. 00 AAAA ATOM 242 HD22 ASN 28 22. 654 35. 468 48. 059 1. 00 0. 00 AAAA ATOM 243 C ASN 28 25. 67636. 670 45. 714 1. 00 24. 12 AAAA ATOM 244 0 ASN 28 26. 063 37. 766 45. 316 1. 00 25. 78 AAAA ATOM 245 N TRP 29 26. 490 35. 759 46. 237 1. 00 22. 87 AAAA ATOM 246 H TRP 29 26. 120 35. 099 46. 851 1. 00 0. 00 AAAA ATOM 247 CA TRP 29 27. 926 35. 739 45. 966 1. 00 21. 65 AAAA ATOM 248 CB TRP 29 28. 737 36. 004 47. 245 1. 00 19. 38 AAAA ATOM 249 CG TRP 29 28. 640 37. 388 47. 766 1. 00 18. 45 AAAA ATOM 250 CD2 TRP 29 29. 44138. 519 47. 389 1. 00 18. 42 AAAA ATOM 251 CE2 TRP 29 28. 946 39. 636 48. 101 1. 00 19. 19 AAAA ATOM 252 CE3 TRP 29 30. 518 38. 706 46. 510 1. 00 19. 40 AAAA ATOM 253 CD1 TRP 29 27. 736 37. 846 48. 672 1. 00 19. 49 AAAA ATOM 254 NE1 TRP 29 27. 909 39. 191 48. 881 1. 00 19. 40 AAAA ATOM 255 HE1 TRP 29 27. 325 39. 692 49. 489 1. 00 0. 00 AAAA ATOM 256 CZ2 TRP 29 29. 487 40. 924 47. 961 1. 00 19. 87 AAAA ATOM 257 CZ3 TRP 29 31. 057 39. 993 46. 367 1. 00 18. 65 AAAA ATOM 258 CH2 TRP 29 30.539 41.079 47.090 1.00 18. 95 AAAA ATOM 259 C TRP 29 28. 233 34. 352 45. 446 1. 00 19. 35 AAAA ATOM 260 0 TRP 29 27. 682 33. 369 45. 953 1. 00 21. 87 AAAA ATOM 261 N GLN 30 29. 000 34. 275 44. 357 1. 00 19. 97 AAAA ATOM 262 H GLN 30 29. 276 35. 096 43. 900 1. 00 0. 00 AAAA ATOM 263 CA GLN 30 29. 401 32. 984 43. 783 1. 00 19. 00 AAAA ATOM 264 CB GLN 30 28. 543 32. 671 42. 552 1. 00 20. 67 AAAA ATOM 265 CG GLN 30 27.042 32.621 42.844 1.00 21. 49 AAAA ATOM 266 CD GLN 30 26.632 31.302 43.408 1.00 20. 18 AAAA ATOM 267 OE1 GLN 30 26. 459 30. 349 42. 671 1. 00 23. 21 AAAA ATOM 268 NE2 GLN 30 26. 63631. 188 44. 725 1. 00 22. 75 AAAA ATOM 269 HE21 GLN 30 26. 874 31. 932 45. 294 1. 00 0. 00 AAAA ATOM 270 HE22 GLN 30 26. 395 30. 274 44. 998 1. 00 0. 00 AAAA ATOM 271 C GLN 30 30. 876 32. 995 43. 383 1. 00 18. 13 AAAA

ATOM 272 O GLN 30 31.378 34.008 42.896 1.00 19. 67 AAAA ATOM 273 N PHE 31 31. 575 31. 892 43. 615 1. 00 18. 15 AAAA ATOM 274 H PHE 31 31. 160 31. 192 44. 148 1. 00 0. 00 AAAA ATOM 275 CA PHE 31 32.952 31.764 43.136 1.00 18. 12 AAAA ATOM 276 CB PHE 31 33. 651 30. 576 43. 791 1. 00 18. 21 AAAA ATOM 277 CG PHE 31 34. 089 30. 841 45. 211 1. 00 21. 78 AAAA ATOM 278 CD1 PHE 31 33. 322 30. 396 46. 288 1. 00 18. 71 AAAA ATOM 279 CD2 PHE 31 35.263 31.563 45.476 1.00 20. 73 AAAA ATOM 280 CE1 PHE 31 33. 719 30. 657 47. 608 1. 00 18. 40 AAAA ATOM 281 CE2 PHE 31 35. 663 31. 826 46. 789 1. 00 17. 15 AAAA ATOM 282 CZ PHE 31 34. 893 31. 374 47. 856 1. 00 16. 61 AAAA ATOM 283 C PHE 31 33. 023 31. 606 41. 631 1. 00 20. 44 AAAA ATOM 284 O PHE 31 32.214 30.891 41.025 1.00 21. 52 AAAA ATOM 285 N VAL 32 34.029 32.232 41.034 1.00 21. 92 AAAA ATOM 286 H VAL 32 34. 545 32. 853 41. 588 1. 00 0. 00 AAAA ATOM 287 CA VAL 32 34. 349 32. 045 39. 616 1. 00 20. 67 AAAA ATOM 288 CB VAL 32 34. 004 33. 332 38. 780 1. 00 20. 87 AAAA ATOM 289 CG1 VAL 32 32. 508 33. 586 38. 787 1. OO 23. 52 AAAA ATOM 290 CG2 VAL 32 34.702 34.563 39.345 1.00 18. 97 AAAA ATOM 291 C VAL 32 35. 847 31. 736 39. 494 1. 00 21. 19 AAAA ATOM 292 0 VAL 32 36. 619 32. 038 40. 405 1. 00 19. 52 AAAA ATOM 293 N ASP 33 36. 251 31. 088 38. 407 1. 00 20. 92 AAAA ATOM 294 H ASP 33 35. 575 30. 751 37. 793 1. 00 0. 00 AAAA ATOM 295 CA ASP 33 37.677 30.936 38.103 1.00 24. 10 AAAA ATOM 296 CB ASP 33 37.877 30.087 36.867 1.00 23. 40 AAAA ATOM 297 CG ASP 33 37. 547 28. 662 37. 101 1. 00 28. 62 AAAA ATOM 298 OD1 ASP 33 38. 155 28. 078 38. 023 1. 00 31. 38 AAAA ATOM 299 OD2 ASP 33 36. 637 28. 146 36. 415 1. 00 31. 16 AAAA ATOM 300 C ASP 33 38.324 32.278 37.849 1.00 24. 54 AAAA ATOM 301 O ASP 33 37.694 33.163 37.268 1.00 25. 36 AAAA ATOM 302 N VAL 34 39. 547 32. 465 38. 333 1. 00 23. 21 AAAA ATOM 303 H VAL 34 39. 885 31. 900 39. 060 1. 00 0. 00 AAAA ATOM 304 CA VAL 34 40. 37933. 527 37. 787 1. 00 26. 15 AAAA ATOM 305 CB VAL 34 41. 176 34. 265 38. 881 1. 00 25. 11 AAAA ATOM 306 CG1 VAL 34 42.030 35.353 38.243 1.00 24. 72 AAAA ATOM 307 CG2 VAL 34 40. 21634. 871 39. 916 1. 00 23. 17 AAAA ATOM 308 C VAL 34 41.324 32.869 36.769 1.00 26. 65 AAAA ATOM 309 0 VAL 34 41. 990 31. 884 37. 080 1. 00 27. 57 AAAA ATOM 310 N TYR 35 41. 149 33. 214 35. 500 1. 00 26. 68 AAAA ATOM 311 H TYR 35 40.563 33.967 35.283 1.00 0. 00 AAAA ATOM 312 CA TYR 35 41. 782 32. 450 34. 430 1. 00 26. 52 AAAA

ATOM 313 CB TYR 35 41. 012 32. 649 33. 116 1. 00 25. 88 AAAA ATOM 314 CG TYR 35 39. 736 31. 831 33. 066 1. 00 22. 69 AAAA ATOM 315 CD1 TYR 35 38. 494 32. 409 33. 344 1. 00 22. 08 AAAA ATOM 316 CE1 TYR 35 37. 348 31. 628 33. 440 1. 00 18. 63 AAAA ATOM 317 CD2 TYR 35 39. 79130. 455 32. 872 1. 00 24. 37 AAAA ATOM 318 CE2 TYR 35 38. 657 29. 672 32. 964 1. 00 23. 29 AAAA ATOM 319 CZ TYR 35 37. 442 30. 264 33. 251 1. 00 21. 25 AAAA ATOM 320 OH TYR 35 36. 321 29. 472 33. 331 1. 00 22. 79 AAAA ATOM 321 HH TYR 35 35. 794 29. 791 34. 083 1. 00 0. 00 AAAA ATOM 322 C TYR 35 43. 262 32. 795 34. 266 1. 00 28. 14 AAAA ATOM 323 0 TYR 35 44. 064 31. 950 33. 869 1. 00 31. 16 AAAA ATOM 324 N GLY 36 43. 615 34. 023 34. 626 1. 00 30. 78 AAAA ATOM 325 H GLY 36 42. 903 34. 661 34. 827 1. 00 0. 00 AAAA ATOM 326 CA GLY 36 45. 013 34. 404 34. 757 1. 00 35. 11 AAAA ATOM 327 C GLY 36 45. 130 35. 712 35. 520 1. 00 37. 27 AAAA ATOM 328 0 GLY 36 44. 114 36. 294 35. 889 1. 00 38. 81 AAAA ATOM 329 N MET 37 46.356 36.177 35.756 1.00 41. 81 AAAA ATOM 330 H MET 37 47. 091 35. 568 35. 539 1. 00 0. 00 AAAA ATOM 331 CA MET 37 46. 590 37. 477 36. 400 1. 00 44. 35 AAAA ATOM 332 CB MET 37 47. 883 37. 457 37. 231 1. 00 48. 91 AAAA ATOM 333 CG MET 37 48. 285 36. 101 37. 820 1. 00 53. 53 AAAA ATOM 334 SD MET 37 47. 107 35. 436 39. 011 1. 00 65. 33 AAAA ATOM 335 CE MET 37 48. 083 34. 161 39. 821 1. 00 58. 45 AAAA ATOM 336 C MET 37 46. 678 38. 598 35. 356 1. 00 45. 24 AAAA ATOM 337 0 MET 37 46. 695 39. 778 35. 694 1. 00 48. 93 AAAA ATOM 338 N ASP 38 46. 841 38. 214 34. 095 1. 00 44. 40 AAAA ATOM 339 H ASP 38 47. 007 37. 262 33. 943 1. 00 0. 00 AAAA ATOM 340 CA ASP 38 46. 75039. 127 32. 958 1. 00 44. 85 AAAA ATOM 341 CB ASP 38 46. 718 38. 296 31. 668 1. 00 54. 39 AAAA ATOM 342 CG ASP 38 47.302 39.024 30.461 1.00 61. 36 AAAA ATOM 343 OD1 ASP 38 47.368 40.273 30.451 1.00 65. 74 AAAA ATOM 344 OD2 ASP 38 47. 647 38. 328 29. 478 1. 00 67. 49 AAAA ATOM 345 C ASP 38 45. 477 39. 980 33. 038 1. 00 42. 01 AAAA ATOM 346 0 ASP 38 44. 382 39. 453 33. 209 1. 00 38. 13 AAAA ATOM 347 N PRO 39 45. 592 41. 284 32. 746 1. 00 42. 09 AAAA ATOM 348 CD PRO 39 46. 871 42. 020 32. 774 1. 00 43. 41 AAAA ATOM 349 CA PRO 39 44. 441 42. 193 32. 631 1. 00 40. 63 AAAA ATOM 350 CB PRO 39 45.046 43.461 32.042 1.00 40. 54 AAAA ATOM 351 CG PRO 39 46.464 43.449 32.509 1.00 42. 45 AAAA ATOM 352 C PRO 39 43. 315 41. 673 31. 744 1. 00 40. 33 AAAA ATOM 353 0 PRO 39 42.16 41.990 31.959 1.00 40. 35 AAAA

ATOM 354 N GLU 40 43. 683 40. 979 30. 675 1. 00 39. 70 AAAA ATOM 355 H GLU 40 44. 637 40. 883 30. 494 1. 00 0. 00 AAAA ATOM 356 CA GLU 40 42. 695 40. 457 29. 736 1. 00 39. 58 AAAA ATOM 357 CB GLU 40 43. 391 39. 907 28. 487 1. 00 38. 40 AAAA ATOM 358 CG GLU 40 43. 806 41. 003 27. 512 1. 00 39. 64 AAAA ATOM 359 CD GLU 40 44. 733 40. 531 26. 388 1. 00 39. 81 AAAA ATOM 360 OE1 GLU 40 45. 443 39. 501 26. 546 1. 00 35. 20 AAAA ATOM 361 OE2 GLU 40 44. 802 41. 261 25. 372 1. 00 37. 81 AAAA ATOM 362 C GLU 40 41. 823 39. 381 30. 377 1. 00 38. 35 AAAA ATOM 363 0 GLU 40 40. 616 39. 318 30. 134 1. 00 39. 28 AAAA ATOM 364 N LEU 41 42. 426 38. 578 31. 248 1. 00 36. 49 AAAA ATOM 365 H LEU 41 43. 38138. 714 31. 409 1. 00 0. 00 AAAA ATOM 366 CA LEU 41 41. 691 37. 538 31. 966 1. 00 35. 44 AAAA ATOM 367 CB LEU 41 42. 567 36. 300 32. 135 1. 00 34. 47 AAAA ATOM 368 CG LEU 41 42. 961 35. 650 30. 805 1. 00 33. 48 AAAA ATOM 369 CD1 LEU 41 44. 058 34. 633 31. 026 1. 00 34. 40 AAAA ATOM 370 CD2 LEU 41 41. 754 35. 002 30. 181 1. 00 26. 42 AAAA ATOM 371 C LEU 41 41. 161 38. 015 33. 321 1. 00 34. 73 AAAA ATOM 372 0 LEU 41 40. 068 37. 623 33. 735 1. 00 35. 09 AAAA ATOM 373 N LEU 42 41. 834 38. 995 33. 918 1. 00 33. 06 AAAA ATOM 374 H LEU 42 42. 629 39. 346 33. 483 1. 00 0. 00 AAAA ATOM 375 CA LEU 42 41. 370 39. 565 35. 177 1. 00 31. 32 AAAA ATOM 376 CB LEU 42 42. 426 40. 495 35. 778 1. 00 31. 53 AAAA ATOM 377 CG LEU 42 43. 141 40. 129 37. 083 1. 00 34. 19 AAAA ATOM 378 CD1 LEU 42 43. 441 41. 416 37. 830 1. 00 37. 43 AAAA ATOM 379 CD2 LEU 42 42.304 39.211 37.950 1.00 33.17 AAAA ATOM 380 C LEU 42 40.074 40.344 34.983 1.00 31. 86 AAAA ATOM 381 0 LEU 42 39. 146 40. 232 35. 782 1. 00 31. 41 AAAA ATOM 382 N SER 43 39. 993 41. 114 33. 905 1. 00 32. 09 AAAA ATOM 383 H SER 43 40. 722 41. 122 33. 244 1. 00 0. 00 AAAA ATOM 384 CA SER 43 38. 809 41. 942 33. 660 1. 00 30. 91 AAAA ATOM 385 CB SER 43 39.067 42.895 32.488 1.00 30. 06 AAAA ATOM 386 OG SER 43 39. 651 42. 207 31. 402 1. 00 33. 39 AAAA ATOM 387 HG SER 43 40. 500 42. 602 31. 162 1. 00 0. 00 AAAA ATOM 388 C SER 43 37.540 41.111 33.401 1.00 27. 31 AAAA ATOM 389 O SER 43 36.430 41.614 33.510 1.00 27. 97 AAAA ATOM 390 N MET 44 37. 709 39. 819 33. 160 1. 00 24. 99 AAAA ATOM 391 H MET 44 38. 618 39. 473 33. 038 1. 00 0. 00 AAAA ATOM 392 CA MET 44 36.570 38.918 33.035 1.00 27. 37 AAAA ATOM 393 CB MET 44 37. 007 37. 600 32. 420 1. 00 29. 97 AAAA ATOM 394 CG MET 44 37. 26537. 623 30. 948 1. 00 30. 45 AAAA

ATOM 395 SD MET 44 38. 047 36. 072 30. 555 1. 00 40. 12 AAAA ATOM 396 CE MET 45 36.647 34.951 30.644 1.00 38. 79 AAAA ATOM 397 C MET 44 35. 838 38. 595 34. 351 1. 00 30. 11 AAAA ATOM 398 0 MET 44 34. 770 37. 981 34. 337 1. 00 31. 53 AAAA ATOM 399 N VAL 45 36. 48438. 830 35. 484 1. 00 29. 44 AAAA ATOM 400 H VAL 45 37. 345 39. 290 35. 448 1. 00 0. 00 AAAA ATOM 401 CA VAL 45 35.893 38.445 36.763 1.00 26. 30 AAAA ATOM 402 CB VAL 45 36. 96538. 411 37. 875 1. 00 24. 98 AAAA ATOM 403 CG1 VAL 45 36. 33838. 051 39. 227 1. 00 21. 57 AAAA ATOM 404 CG2 VAL 45 38.043 37.412 37.503 1.00 23. 63 AAAA ATOM 405 C VAL 45 34.747 39.381 37.173 1.00 26. 01 AAAA ATOM 406 0 VAL 45 34. 90140. 603 37. 199 1. 00 25. 56 AAAA ATOM 407 N PRO 46 33. 566 38. 820 37. 457 1. 00 26. 96 AAAA ATOM 408 CD PRO 46 33.174 37.412 37.264 1.00 28. 85 AAAA ATOM 409 CA PRO 46 32. 446 39. 616 37. 959 1. 00 26. 67 AAAA ATOM 410 CB PRO 46 31.356 38.578 38.209 1.00 26. 01 AAAA ATOM 411 CG PRO 46 31. 679 37. 474 37. 282 1. 00 24. 45 AAAA ATOM 412 C PRO 46 32. 822 40. 338 39. 234 1. 00 26. 63 AAAA ATOM 413 0 PRO 46 33. 536 39. 788 40. 054 1. 00 29. 14 AAAA ATOM 414 N ARG 47 32.427 41.600 39.348 1.00 28. 89 AAAA ATOM 415 H ARG 47 31. 947 41. 987 38. 584 1. 00 0. 00 AAAA ATOM 416 CA ARG 47 32.660 42.390 40.558 1.00 31. 96 AAAA ATOM 417 CB ARG 47 33. 439 43. 660 40. 232 1. 00 38. 74 AAAA ATOM 418 CG ARG 47 34. 628 43. 440 39. 357 1. 00 45. 55 AAAA ATOM 419 CD ARG 47 35.882 43.853 40.069 1.00 53. 22 AAAA ATOM 420 NE ARG 47 37. 031 43. 644 39. 196 1. 00 57. 19 AAAA ATOM 421 HE ARG 47 36. 970 42. 863 38. 611 1. 00 0. 00 AAAA ATOM 422 CZ ARG 47 38.126 44.395 39.204 1.00 59. 13 AAAA ATOM 423 NH1 ARG 47 39. 033 44. 235 38. 246 1. 00 58. 34 AAAA ATOM 424 HHll ARG 47 38. 884 43. 574 37. 513 1. 00 0. 00 AAAA ATOM 425 HH12ARG 47 39. 848 44. 815 38. 243 1. 00 0. 00 AAAA ATOM 426 NH2 ARG 47 38. 341 45. 252 40. 202 1. 00 57. 19 AAAA ATOM 427 HH21 ARG 47 39. 161 45. 822 40. 207 1. 00 0. 00 AAAA ATOM 428 HH22ARG 47 37. 667 45. 360 40. 937 1. 00 0. 00 AAAA ATOM 429 C ARG 47 31.334 42.788 41.207 1.00 31. 57 AAAA ATOM 430 0 ARG 47 30.279 42.698 40.578 1.00 35. 91 AAAA ATOM 431 N PRO 48 31. 375 43. 285 42. 450 1. 00 28. 07 AAAA ATOM 432 CD PRO 48 30.207 43.903 43.087 1.00 29. 27 AAAA ATOM 433 CA PRO 48 32. 511 43. 267 43. 374 1. 00 27. 18 AAAA ATOM 434 CB PRO 48 31. 948 43. 916 44. 635 1. 00 25. 16 AAAA ATOM 435 CG PRO 48 30.837 44.729 44.159 1.00 30. 03 AAAA

ATOM 436 C PRO 48 33. 010 41. 854 43. 664 1. 00 26. 56 AAAA ATOM 437 0 PRO 48 32. 286 40. 875 43. 454 1. 00 24. 00 AAAA ATOM 438 N VAL 49 34. 301 41. 761 43. 982 1. 00 25. 54 AAAA ATOM 439 H VAL 49 34. 852 42. 554 43. 813 1. 00 0. 00 AAAA ATOM 440 CA VAL 49 34. 91140. 547 44. 520 1. 00 21. 73 AAAA ATOM 441 CB VAL 49 36.246 40.261 43.799 1.00 22. 26 AAAA ATOM 442 CG1 VAL 49 36. 948 39. 064 44. 423 1. 00 20. 99 AAAA ATOM 443 CG2 VAL 49 35. 991 40. 004 42. 332 1. 00 21. 69 AAAA ATOM 444 C VAL 49 35. 18240. 791 46. 004 1. 00 17. 83 AAAA ATOM 445 0 VAL 49 35. 671 41. 851 46. 363 1. 00 19. 33 AAAA ATOM 446 N CYS 50 34. 736 39. 895 46. 880 1. 00 19. 34 AAAA ATOM 447 H CYS 50 34. 184 39. 155 46. 538 1. 00 0. 00 AAAA ATOM 448 CA CYS 50 35.030 40.082 48.305 1.00 19. 43 AAAA ATOM 449 CB CYS 50 33. 75140. 098 49. 147 1. 00 18. 28 AAAA ATOM 450 SG CYS 50 32. 923 38. 524 49. 247 1. 00 22. 22 AAAA ATOM 451 C CYS 50 36. 016 39. 068 48. 892 1. 00 18. 64 AAAA ATOM 452 0 CYS 50 36.363 39.141 50.067 1.00 21. 41 AAAA ATOM 453 N ALA 51 36.527 38.174 48.064 1.00 16. 97 AAAA ATOM 454 H ALA 51 36. 204 38. 132 47. 146 1. 00 0. 00 AAAA ATOM 455 CA ALA 51 37. 527 37. 235 48. 525 1. 00 12. 98 AAAA ATOM 456 CB ALA 51 36. 899 36. 197 49. 434 1. 00 15. 92 AAAA ATOM 457 C ALA 51 38. 156 36. 559 47. 345 1. 00 17. 22 AAAA ATOM 458 0 ALA 51 37. 489 36. 264 46. 361 1. 00 18. 29 AAAA ATOM 459 N VAL 52 39. 454 36. 304 47. 447 1. 00 16. 94 AAAA ATOM 460 H VAL 52 39. 935 36. 706 48. 201 1. 00 0. 00 AAAA ATOM 461 CA VAL 52 40. 148 35. 446 46. 485 1. 00 16. 89 AAAA ATOM 462 CB VAL 52 41.325 36.226 45.769 1.00 14. 71 AAAA ATOM 463CG1VAL5242. 10435. 29544. 8641. 0014. 13 AAAA ATOM 464 CG2 VAL 52 40. 784 37. 417 44. 983 1. 00 15. 31 AAAA ATOM 465 C VAL 52 40. 711 34. 245 47. 249 1. 00 15. 15 AAAA ATOM 466 0 VAL 52 41. 310 34. 403 48. 326 1. 00 18. 19 AAAA ATOM 467 N LEU 53 40.428 33.050 46.765 1.00 12. 59 AAAA ATOM 468 H LEU 53 39. 784 32. 985 46. 031 1. 00 0. 00 AAAA ATOM 469 CA LEU 53 41. 081 31. 873 47. 287 1. 00 14. 74 AAAA ATOM 470 CB LEU 53 40. 067 30. 754 47. 519 1. 00 16. 32 AAAA ATOM 471 CG LEU 53 39. 487 30. 583 48. 937 1. 00 18. 05 AAAA ATOM 472 CD1 LEU 53 38. 98631. 897 49. 449 1. 00 16. 19 AAAA ATOM 473 CD2 LEU 53 38. 370 29. 542 48. 921 1. 00 15. 85 AAAA ATOM 474 C LEU 53 42.186 31.408 46.327 1.00 20. 78 AAAA ATOM 475 0 LEU 53 41.992 31.395 45.109 1.00 15. 78 AAAA ATOM 476 N LEU 54 43.389 31.205 46.873 1.00 19. 44 AAAA

ATOM 477 H LEU 54 43. 473 31. 356 47. 838 1. 00 0. 00 AAAA ATOM 478 CA LEU 54 44.527 30.741 46.081 1.00 19. 71 AAAA ATOM 479 CB LEU 54 45. 728 31. 698 46. 209 1. 00 19. 22 AAAA ATOM 480 CG LEU 54 47. 039 31. 318 45. 501 1. 00 18. 95 AAAA ATOM 481 CD1 LEU 54 46.8563 31.342 43.981 1.00 15. 60 AAAA ATOM 482 CD2 LEU 54 48.118 32.309 45.904 1.00 18. 59 AAAA ATOM 483 C LEU 54 44. 938 29. 353 46. 504 1. 00 19. 65 AAAA ATOM 484 0 LEU 54 45. 116 29. 081 47. 709 1. 00 16. 10 AAAA ATOM 485 N LEU 55 44. 939 28. 457 45. 517 1. 00 19. 34 AAAA ATOM 486 H LEU 55 44. 559 28. 742 44. 656 1. 00 0. 00 AAAA ATOM 487 CA LEU 55 45. 465 27. 098 45. 662 1. 00 19. 42 AAAA ATOM 488 CB LEU 55 44. 586 26. 099 44. 908 1. 00 18. 64 AAAA ATOM 489 CG LEU 55 44. 979 24. 625 45. 027 1. 00 20. 11 AAAA ATOM 490 CD1 LEU 55 44. 716 24. 084 46. 435 1. 00 22. 00 AAAA ATOM 491 CD2 LEU 55 44. 183 23. 827 44. 019 1. 00 22. 10 AAAA ATOM 492 C LEU 55 46.882 27.044 45.105 1.00 22. 01 AAAA ATOM 493 0 LEU 55 47. 107 27. 348 43. 930 1. 00 19. 66 AAAA ATOM 494 N PHE 56 47. 836 26. 685 45. 959 1. 00 21. 06 AAAA ATOM 495 H PHE 56 47. 572 26. 417 46. 861 1. 00 0. 00 AAAA ATOM 496 CA PHE 56 49. 249 26. 696 45. 580 1. 00 20. 59 AAAA ATOM 497 CB PHE 56 49. 883 28. 048 45. 948 1. 00 16. 88 AAAA ATOM 498 CG PHE 56 50. 061 28. 261 47. 418 1. 00 23. 60 AAAA ATOM 499 CD1 PHE 56 51. 221 27. 831 48. 063 l. 00 25. 11 AAAA ATOM 500 CD2 PHE 56 49. 073 28. 886 48. 172 1. 00 23. 92 AAAA ATOM 501 CE1 PHE 56 51. 384 28. 015 49. 433 1. 00 27. 68 AAAA ATOM 502 CE2 PHE 56 49. 235 29. 078 49. 549 1. 00 23. 11 AAAA ATOM 503 CZ PHE 56 50. 383 28. 642 50. 176 1. 00 26. 70 AAAA ATOM 504 C PHE 56 49. 963 25. 540 46. 266 1. 00 22. 28 AAAA ATOM 505 0 PHE 56 49. 394 24. 890 47. 158 1. 00 21. 61 AAAA ATOM 506 N PRO 57 51. 193 25. 208 45. 819 1. 00 27. 77 AAAA ATOM 507 CD PRO 57 51. 930 25. 733 44. 651 1. 00 25. 94 AAAA ATOM 508 CA PRO 57 51. 891 24. 060 46. 423 1. 00 26. 56 AAAA ATOM 509 CB PRO 57 52.538 23.377 45.216 1.00 27. 54 AAAA ATOM 510 CG PRO 57 52.668 24.507 44.160 1.00 29. 50 AAAA ATOM 511 C PRO 57 52.906 24.439 47.523 1.00 23. 83 AAAA ATOM 512 0 PRO 57 53. 565 25. 481 47. 460 1. 00 23. 72 AAAA ATOM 513 N ILE 58 52.874 23.689 48.615 1.00 27. 04 AAAA ATOM 514 H ILE 58 52. 184 23. 055 48. 691 1. 00 0. 00 AAAA ATOM 515 CA ILE 58 53. 800 23. 906 49. 728 1. 00 31. 09 AAAA ATOM 516 CB ILE 58 53. 241 23. 338 51. 064 1. 00 32. 61 AAAA ATOM 517 CG2 ILE 58 54. 204 23. 642 52. 226 1. 00 31. 31 AAAA

ATOM 518 CG1 ILE 58 51. 875 23. 949 51. 363 1. 00 30. 84 AAAA ATOM 519 CD ILE 58 51. 174 23. 250 52. 487 1. 00 29. 57 AAAA ATOM 520 C ILE 58 55.142 23.234 49.427 1.00 31. 74 AAAA ATOM 521 0 ILE 58 55. 278 22. 009 49. 512 1. 00 33. 47 AAAA ATOM 522 N THR 59 56. 072 24. 022 48. 910 1. 00 33. 63 AAAA ATOM 523 H THR 59 55. 804 24. 955 48. 785 1. 00 0. 00 AAAA ATOM 524 CA THR 59 57. 390 23. 511 48. 556 1. 00 32. 86 AAAA ATOM 525 CB THR 59 57. 838 24. 082 47. 205 1. 00 34. 38 AAAA ATOM 526 OG1 THR 59 57. 86425. 513 47. 284 1. 00 30. 05 AAAA ATOM 527 HG1 THR 59 57. 104 25. 829 46. 781 1. 00 0. 00 AAAA ATOM 528 CG2 THR 59 56. 877 23. 658 46. 091 1. 00 34. 44 AAAA ATOM 529 C THR 59 58. 417 23. 901 49. 616 1. 00 34. 06 AAAA ATOM 530 0 THR 59 58. 157 24. 786 50. 429 1. 00 30. 95 AAAA ATOM 531 N GLU 60 59. 593 23. 271 49. 578 1. 00 36. 00 AAAA ATOM 532 H GLU 60 59. 653 22. 488 48. 995 1. 00 0. 00 AAAA ATOM 533 CA GLU 60 60. 720 23. 670 50. 426 1. 00 36. 26 AAAA ATOM 534 CB GLU 60 61. 980 22. 903 50. 033 1. 00 42. 18 AAAA ATOM 535 CG GLU 60 62. 050 21. 471 50. 549 1. 00 53. 87 AAAA ATOM 536 CD GLU 60 63. 29620. 736 50. 068 1. 00 59. 16 AAAA ATOM 537 OE1 GLU 60 64. 348 20. 813 50. 747 1. 00 63. 06 AAAA ATOM 538 OE2 GLU 60 63. 225 20. 091 48. 996 1. 00 63. 28 AAAA ATOM 539 C GLU 60 60. 98225. 153 50. 256 1. 00 35. 56 AAAA ATOM 540 0 GLU 60 61.101 25.890 51.228 1.00 37. 03 AAAA ATOM 541 N LYS 61 60. 986 25. 582 48. 999 1. 00 35. 28 AAAA ATOM 542 H LYS 61 60. 883 24. 902 48. 307 1. 00 0. 00 AAAA ATOM 543 CA LYS 61 61. 178 26. 974 48. 607 1. 00 35. 78 AAAA ATOM 544 CB LYS 61 61. 079 27. 060 47. 088 1. 00 38. 62 AAAA ATOM 545 CG LYS 61 61. 833 28. 185 46. 448 1. 00 43. 95 AAAA ATOM 546 CD LYS 61 62. 080 27. 843 44. 990 1. 00 46. 20 AAAA ATOM 547 CE LYS 61 63. 096 28. 769 44. 355 1. 00 48. 83 AAAA ATOM 548 NZ LYS 61 63. 535 28. 244 43. 029 1. 00 51. 71 AAAA ATOM 549 HZ1 LYS 61 62. 705 28. 054 42. 432 1. 00 0. 00 AAAA ATOM 550 HZ2 LYS 61 64. 068 27. 364 43. 181 1. 00 0. 00 AAAA ATOM 551 HZ3 LYS 61 64. 154 28. 942 42. 568 1. 00 0. 00 AAAA ATOM 552 C LYS 61 60. 115 27. 877 49. 246 1. 00 36. 67 AAAA ATOM 553 0 LYS 61 60. 425 28. 913 49. 836 1. 00 36. 93 AAAA ATOM 554 N TYR 62 58.859 27.447 49.168 1.00 35. 28 AAAA ATOM 555 H TYR 62 58. 677 26. 650 48. 629 1. 00 0. 00 AAAA ATOM 556 CA TYR 62 57. 764 28. 114 49. 866 1. 00 31. 76 AAAA ATOM 557 CB TYR 62 56. 460 27. 342 49. 626 1. 00 31. 61 AAAA ATOM 558 CG TYR 62 55. 310 27. 834 50. 461 1. 00 26. 21 AAAA

ATOM 559 CD1 TYR 62 54. 761 29. 088 50. 222 1. 00 30. 35 AAAA ATOM 560 CE1 TYR 62 53. 856 29. 659 51. 106 1. 00 29. 55 AAAA ATOM 561 CD2 TYR 62 54. 905 27. 144 51. 600 1. 00 25. 46 AAAA ATOM 562 CE2 TYR 62 54. 001 27. 710 52. 501 1. 00 25. 74 AAAA ATOM 563 CZ TYR 62 53. 488 28. 967 52. 243 1. 00 26. 36 AAAA ATOM 564 OH TYR 62 52. 641 29. 589 53. 116 1. 00 26. 54 AAAA ATOM 565 HH TYR 62 52. 044 30. 083 52. 548 1. 00 0. 00 AAAA ATOM 566 C TYR 62 58. 059 28. 190 51. 372 1. 00 30. 10 AAAA ATOM 567 0 TYR 62 58. 032 29. 266 51. 976 1. 00 31. 64 AAAA ATOM 568 N GLU 63 58. 434 27. 054 51. 946 1. 00 31. 85 AAAA ATOM 569 H GLU 63 58. 606 26. 285 51. 371 1. 00 0. 00 AAAA ATOM 570 CA GLU 63 58. 638 26. 941 53. 387 1. 00 34. 36 AAAA ATOM 571 CB GLU 63 58. 892 25. 488 53. 770 1. 00 35. 57 AAAA ATOM 572 CG GLU 63 57. 659 24. 624 53. 771 1. 00 36. 98 AAAA ATOM 573 CD GLU 63 56. 658 25. 070 54. 806 1. 00 38. 63 AAAA ATOM 574 OE1 GLU 63 55. 884 26. 008 54. 529 1. 00 38. 48 AAAA ATOM 575 OE2 GLU 63 56. 652 24. 479 55. 900 1. 00 41. 70 AAAA ATOM 576 C GLU 63 59. 759 27. 814 53. 960 1. 00 35. 89 AAAA ATOM 577 0 GLU 63 59. 650 28. 290 55. 099 1. 00 35. 16 AAAA ATOM 578 N IIAL 64 60. 835 28. 035 53. 207 1. 00 34. 07 AAAA ATOM 579 H VAL 64 60. 904 27. 569 52. 341 1. 00 0. 00 AAAA ATOM 580 CA VAL 64 61. 879 28. 901 53. 744 1. 00 32. 48 AAAA ATOM 581 CB VAL 64 63. 282 28. 790 52. 994 1. 00 34. 25 AAAA ATOM 582 CG1 VAL 64 63.535 27.365 52.550 1.00 30. 44 AAAA ATOM 583 CG2 VAL 64 63. 400 29. 793 51. 831 1. 00 32. 48 AAAA ATOM 584 C VAL 64 61. 37330. 339 53. 768 1. 00 30. 07 AAAA ATOM 585 0 VAL 64 61. 497 31. 023 54. 794 1. 00 30. 66 AAAA ATOM 586 N PHE 65 60.649 30.739 52.728 1.00 25. 14 AAAA ATOM 587 H PHE 65 60. 486 30. 125 51. 980 1. 00 0. 00 AAAA ATOM 588 CA PHE 65 60. 092 32. 077 52. 726 1. 00 27. 03 AAAA ATOM 589 CB PHE 65 59.362 32.379 51.424 1.00 28. 76 AAAA ATOM 590 CG PHE 65 58.726 33.738 51.403 1.00 30. 67 AAAA ATOM 591 CD1 PHE 65 59. 449 34. 847 50. 985 1. 00 31. 43 AAAA ATOM 592 CD2 PHE 65 57. 462 33. 930 51. 945 1. 00 31. 83 AAAA ATOM 593 CE1 PHE 65 58. 933 36. 127 51. 122 1. 00 33. 66 AAAA ATOM 594 CE2 PHE 65 56.940 35.203 52.090 1.00 31. 50 AAAA AT OM 595 CZ PHE 65 57. 677 36. 306 51. 681 1. 00 34. 79 AAAA ATOM 596 C PHE 65 59.127 32.255 53.898 1.00 27. 51 AAAA ATOM 597 0 PHE 65 59. 178 33. 253 54. 602 1. 00 28. 94 AAAA ATOM 598 N ARG 66 58. 233 31. 294 54. 087 1. 00 29. 57 AAAA ATOM 599 H ARG 66 58. 21130. 557 53. 439 1. 00 0. 00 AAAA

ATOM 600 CA ARG 66 57. 277 31. 358 55. 189 1. 00 28. 53 AAAA ATOM 601 CB ARG 66 56. 409 30. 103 55. 210 1. 00 28. 27 AAAA ATOM 602 CG ARG 66 55. 210 30. 274 56. 099 1. 00 30. 07 AAAA ATOM 603 CD ARG 66 54.586 28.967 56.414 1.00 34. 44 AAAA ATOM 604 NE ARG 66 55. 118 28. 447 57. 657 1. 00 43. 87 AAAA ATOM 605 HE ARG 66 55. 420 29. 082 58. 336 1. 00 0. 00 AAAA ATOM 606 CZ ARG 66 55. 251 27. 159 57. 915 1. 00 47. 87 AAAA ATOM 607 NH1 ARG 66 55. 743 26. 760 59. 077 1. 00 56. 13 AAAA ATOM 608 HH11 ARG 66 55. 911 27. 432 59. 798 1. 00 0. 00 AAAA ATOM 609 HH12ARG 66 55. 849 25. 785 59. 271 1. 00 0. 00 AAAA ATOM 610 NH2 ARG 66 54. 831 26. 267 57. 034 1. 00 54. 07 AAAA ATOM 611 HH21 ARG 66 54. 902 25. 291 57. 249 1. 00 0. 00 AAAA ATOM 612 HH22ARG 66 54. 365 26. 557 56. 200 1. 00 0. 00 AAAA ATOM 613 C ARG 66 57. 923 31. 562 56. 574 1. 00 28. 57 AAAA ATOM 614 0 ARG 66 57. 495 32. 431 57. 349 1. 00 26. 47 AAAA ATOM 615 N THR 67 58. 993 30. 817 56. 856 1. 00 28. 49 AAAA ATOM 616 H THR 67 59. 242 30. 128 56. 207 1. 00 0. 00 AAAA ATOM 617 CA THR 67 59. 711 30. 970 58. 127 1. 00 26. 81 AAAA ATOM 618 CB THR 67 60. 732 29. 895 58. 333 1. 00 27. 83 AAAA ATOM 619 OG1 THR 67 60. 144 28. 631 58. 024 1. 00 37. 76 AAAA ATOM 620 HG1 THR 67 60.091 28.441 57.077 1.00 0. 00 AAAA ATOM 621 CG2 THR 67 61. 157 29. 879 59. 784 1. 00 34. 84 AAAA ATOM 622 C THR 67 60. 425 32. 297 58. 270 1. 00 24. 31 AAAA ATOM 623 0 THR 67 60. 343 32. 931 59. 316 1. 00 25. 88 AAAA ATOM 624 N GLU 68 61. 016 32. 776 57. 180 1. 00 24. 39 AAAA ATOM 625 H GLU 68 61. 054 32. 210 56. 386 1. 00 0. 00 AAAA ATOM 626 CA GLU 68 61. 576 34. 117 57. 165 1. 00 26. 47 AAAA ATOM 627 CB GLU 68 62. 239 34. 391 55. 817 1. 00 25. 86 AAAA ATOM 628 CG GLU 68 63. 442 33. 483 55. 597 1. 00 34. 86 AAAA ATOM 629 CD GLU 68 64. 410 33. 971 54. 528 1. 00 32. 64 AAAA ATOM 630 OE1 GLU 68 64. 606 35. 207 54. 393 1. 00 33. 07 AAAA ATOM 631 OE2 GLU 68 65. 006 33. 097 53. 862 1. 00 30. 30 AAAA ATOM 632 C GLU 68 60. 509 35. 174 57. 451 1. 00 28. 99 AAAA ATOM 633 0 GLU 68 60.692 36.049 58.309 1.00 27. 30 AAAA ATOM 634 N GLU 69 59. 368 35. 048 56. 771 1. 00 28. 12 AAAA ATOM 635 H GLU 69 59. 312 34. 308 56. 134 1. 00 0. 00 AAAA ATOM 636 CA GLU 69 58. 24135. 966 56. 952 1. 00 24. 58 AAAA ATOM 637 CB GLU 69 57. 096 35. 565 56. 017 1. 00 23. 71 AAAA ATOM 638 CG GLU 69 55. 847 36. 368 56. 196 1. 00 22. 11 AAAA ATOM 639 CD GLU 69 54. 70535. 813 55. 380 1. 00 25. 45 AAAA ATOM 640 OE1 GLU 69 54. 228 34. 710 55. 713 1. 00 23. 09 AAAA

ATOM 641 OE2 GLU 69 54. 296 36. 476 54. 404 1. 00 26. 44 AAAA ATOM 642 C GLU 69 57. 754 35. 984 58. 404 1. 00 21. 08 AAAA ATOM 643 0 GLU 69 57. 476 37. 041 58. 959 1. 00 20. 51 AAAA ATOM 644 N GLU 70 57. 714 34. 814 59. 028 1. 00 19. 93 AAAA ATOM 645 H GLU 70 57.946 34.008 58.528 1.00 0. 00 AAAA ATOM 646 CA GLU 70 57. 309 34. 721 60. 419 1. 00 24. 42 AAAA ATOM 647 CB GLU 70 57. 136 33. 256 60. 815 1. 00 24. 09 AAAA ATOM 648 CG GLU 70 56. 382 33. 079 62. 114 1. 00 25. 29 AAAA ATOM 649 CD GLU 70 56. 333 31. 650 62. 584 1. 00 24. 10 AAAA ATOM 650 OE1 GLU 70 56. 101 30. 753 61. 745 1. 00 25. 29 AAAA ATOM 651 OE2 GLU 70 56. 489 31. 431 63. 806 1. 00 26. 77 AAAA ATOM 652 C GLU 70 58. 325 35. 387 61. 354 1. 00 26. 36 AAAA ATOM 653 0 GLU 70 57. 959 36. 181 62. 222 1. 00 27. 60 AAAA ATOM 654 N GLU 71 59. 605 35. 153 61. 091 1. 00 28. 74 AAAA ATOM 655 H GLU 71 59. 819 34. 531 60. 361 1. 00 0. 00 AAAA ATOM 656 CA GLU 71 60. 684 35. 764 61. 873 1. 00 31. 06 AAAA ATOM 657 CB GLU 71 62. 027 35. 154 61. 463 1. 00 34. 77 AAAA ATOM 658 CG GLU 71 62. 138 33. 657 61. 762 1. 00 45. 41 AAAA ATOM 659 CD GLU 71 63. 521 33. 074 61. 448 1. 00 56. 76 AAAA ATOM 660 OE1 GLU 71 64. 276 33. 686 60. 642 1. 00 58. 43 AAAA ATOM 661 OE2 GLU 71 63. 846 31. 993 62. 008 1. 00 59. 87 AAAA ATOM 662 C GLU 71 60. 724 37. 289 61. 717 1. 00 27. 50 AAAA ATOM 663 0 GLU 71 60. 776 38. 029 62. 706 1. 00 26. 56 AAAA ATOM 664 N LYS 72 60. 557 37. 752 60. 485 1. 00 24. 83 AAAA ATOM 665 H LYS 72 60. 474 37. 104 59. 763 1. 00 0. 00 AAAA ATOM 666 CA LYS 72 60. 517 39. 184 60. 216 1. 00 27. 68 AAAA ATOM 667 CB LYS 72 60. 501 39. 444 58. 704 1. 00 29. 44 AAAA ATOM 668 CG LYS 72 60. 63440. 917 58. 346 1. 00 35. 91 AAAA ATOM 669 CD LYS 72 60. 541 41. 141 56. 849 1. 00 45. 30 AAAA ATOM 670 CE LYS 72 59.768 42.427 56.528 1.00 50. 61 AAAA ATOM 671 NZ LYS 72 58. 357 42. 379 57. 040 1. 00 52. 85 AAAA ATOM 672 HZ1 LYS 72 58. 363 42. 253 58. 072 1. 00 0. 00 AAAA ATOM 673 HZ2 LYS 72 57. 867 41. 574 56. 600 1. 00 0. 00 AAAA ATOM 674 HZ3 LYS 72 57. 864 43. 263 56. 803 1. 00 0. 00 AAAA ATOM 675 C LYS 72 59. 336 39. 904 60. 898 1. 00 28. 50 AAAA ATOM 676 0 LYS 72 59.505 40.990 61.453 1.00 26. 07 AAAA ATOM 677 N ILE 73 58. 16339. 269 60. 925 1. 00 27. 98 AAAA ATOM 678 H ILE 73 58. 085 38. 402 60. 469 1. 00 0. 00 AAAA ATOM 679 CA ILE 73 57. 014 39. 870 61. 586 1. 00 30. 16 AAAA ATOM 680 CB ILE 73 55. 678 39. 221 61. 117 1. 00 32. 09 AAAA ATOM 681 CG2 ILE 73 54. 518 39. 615 62. 075 1. 00 28. 61 AAAA

ATOM 682 CG1 ILE 73 55. 362 39. 681 59. 683 1. 00 30. 18 AAAA ATOM 683 CD ILE 73 54. 303 38. 839 58. 964 1. 00 28. 53 AAAA ATOM 684 C ILE 73 57. 117 39. 813 63. 117 1. 00 32. 56 AAAA ATOM 685 0 ILE 73 56.658 40.716 63.817 1.00 31. 86 AAAA ATOM 686 N LYS 74 57. 758 38. 778 63. 637 1. 00 34. 68 AAAA ATOM 687 H LYS 74 58. 046 38. 050 63. 047 1. 00 0. 00 AAAA ATOM 688 CA LYS 74 57. 996 38. 703 65. 070 1. 00 38. 56 AAAA ATOM 689 CB LYS 74 58. 455 37. 292 65. 439 1. 00 41. 67 AAAA ATOM 690 CG LYS 74 57. 374 36. 221 65. 241 1. 00 44. 95 AAAA ATOM 691 CD LYS 74 57. 997 34. 832 65. 096 1. 00 49. 48 AAAA ATOM 692 CE LYS 74 57. 330 33. 811 66. 003 1. 00 50. 67 AAAA ATOM 693 NZ LYS 74 55. 898 33. 585 65. 661 1. 00 53. 91 AAAA ATOM 694 HZl LYS 74 55. 817 32. 986 64. 817 1. 00 0. 00 AAAA ATOM 695 HZ2 LYS 74 55. 437 34. 499 65. 477 1. 00 0. 00 AAAA ATOM 696 HZ3 LYS 74 55.431 33.117 66.465 1.00 0. 00 AAAA ATOM 697 C LYS 74 59. 014 39. 764 65. 542 1. 00 40. 96 AAAA ATOM 698 0 LYS 74 58.880 40.327 66.627 1.00 41. 57 AAAA ATOM 699 N SER 75 59. 955 40. 122 64. 675 1. 00 41. 35 AAAA ATOM 700 H SER 75 60. 035 39. 595 63. 850 1. 00 0. 00 AAAA ATOM 701 CA SER 75 60.878 41.219 64.969 1.00 43. 19 AAAA ATOM 702 CB SER 75 62. 102 41. 163 64. 046 1. 00 44. 46 AAAA ATOM 703 OG SER 75 62.467 42.463 63.571 1.00 46. 60 AAAA ATOM 704 HG SER 75 62. 724 43. 054 64. 265 1. 00 0. 00 AAAA ATOM 705 C SER 75 60.217 42.587 64.828 1.00 44. 73 AAAA ATOM 706 0 SER 75 60. 382 43. 463 65. 685 1. 00 48. 52 AAAA ATOM 707 N GLN 76 59. 518 42. 785 63. 715 1. 00 41. 15 AAAA ATOM 708 H GLN 76 59. 362 42. 019 63. 126 1. 00 0. 00 AAAA ATOM 709 CA GLN 76 59.108 44.117 63.303 1.00 38. 47 AAAA ATOM 710 CB GLN 76 59. 590 44. 350 61. 875 1. 00 41. 98 AAAA ATOM 711 CG GLN 76 58.516 44.515 60.824 1.00 43. 50 AAAA ATOM 712 CD GLN 76 59. 007 45. 325 59. 660 1. 00 44. 41 AAAA ATOM 713 OE1 GLN 76 60. 11945. 841 59. 675 1. 00 43. 52 AAAA ATOM 714 NE2 GLN 76 58. 182 45. 449 58. 644 1. 00 48. 56 AAAA ATOM 715 HE21 GLN 76 57.294 45.040 58.703 1.00 0. 00 AAAA ATOM 716 HE22 GLN 76 58. 569 45. 963 57. 911 1. 00 0. 00 AAAA ATOM 717 C GLN 76 57. 606 44. 411 63. 444 1. 00 35. 54 AAAA ATOM 718 0 GLN 76 57. 170 4. 564 63. 305 1. 00 33. 94 AAAA ATOM 719 N GLY 77 56.852 43.400 63.867 1.00 31. 89 AAAA ATOM 720 H GLY 77 57. 272 42. 529 63. 999 1. 00 0. 00 AAAA ATOM 721 CA GLY 7J 55. 42943. 573 64. 095 1. 00 29. 13 AAAA ATOM 722 C GLY 77 54. 617 43. 816 62. 833 1. 00 26. 74 AAAA

ATOM 723 0 GLY 77 55. 167 44. 016 61. 743 1. 00 25. 60 AAAA ATOM 724 N GLN 78 53. 297 43. 804 62. 985 1. 00 24. 43 AAAA ATOM 725 H GLN 78 52.921 43.551 63.845 1.00 0. 00 AAAA ATOM 726 CA GLN 78 52.385 44.167 61.900 1.00 22. 99 AAAA ATOM 727 CB GLN 78 52.217 42.990 60.942 1.00 23. 16 AAAA ATOM 728 CG GLN 78 51. 534 41. 796 61. 602 1. 00 23. 53 AAAA ATOM 729 CD GLN 78 51.099 40.730 60.627 1.00 17. 76 AAAA ATOM 730 OE1 GLN 78 51.185 40.900 59.408 1.00 16. 41 AAAA ATOM 731 NE2 GLN 78 50. 685 39. 597 61. 160 1. 00 15. 76 AAAA ATOM 732 HE21 GLN 78 50. 651 39. 510 62. 138 1. 00 0. 00 AAAA ATOM 733 HE22 GLN 78 50. 447 38. 904 60. 516 1. 00 0. 00 AAAA ATOM 734 C GLN 78 51. 032 44. 498 62. 540 1. 00 25. 68 AAAA ATOM 735 0 GLN 78 50.757 44.098 63.676 1.00 26. 20 AAAA ATOM 736 N ASP 79 50.185 45.223 61.826 1.00 23. 31 AAAA ATOM 737 H ASP 79 50. 483 45. 471 60. 932 1. 00 0. 00 AAAA ATOM 738 CA ASP 79 48. 838 45. 466 62. 326 1. 00 25. 37 AAAA ATOM 739 CB ASP 79 48. 386 46. 880 61. 962 1. 00 24. 45 AAAA ATOM 740 CG ASP 79 49. 211 47. 959 62. 672 1. 00 28. 21 AAAA ATOM 741 OD1 ASP 79 50. 071 47. 598 63. 516 1. 00 26. 57 AAAA ATOM 742 OD2 ASP 79 49. 018 49. 158 62. 360 1. 00 28. 96 AAAA ATOM 743 C ASP 79 47.824 44.428 61.824 1.00 27. 86 AAAA ATOM 744 0 ASP 79 47. 821 44. 089 60. 638 1. 00 26. 79 AAAA ATOM 745 N VAL 80 47. 133 43. 784 62. 770 1. 00 27. 61 AAAA ATOM 746 H VAL 80 47. 439 43. 860 63. 690 1. 00 0. 00 AAAA ATOM 747 CA VAL 80 45. 971 42. 948 62. 457 1. 00 25. 15 AAAA ATOM 748 CB VAL 80 46. 207 41. 477 62. 840 1. 00 23. 30 AAAA ATOM 749 CG1 VAL 80 45. 00740. 651 62. 450 1. 00 25. 36 AAAA ATOM 750 CG2 VAL 80 47. 438 40. 941 62. 141 1. 00 22. 16 AAAA ATOM 751 C VAL 80 44.739 44.452 63.201 1.00 24. 57 AAAA ATOM 752 0 VAL 80 44. 644 43. 295 64. 413 1. 00 22. 28 AAAA ATOM 753 N THR 81 43. 813 44. 073 62. 474 1. 00 24. 26 AAAA ATOM 754 H THR 81 44. 032 44. 168 61. 520 1. 00 0. 00 AAAA ATOM 755 CA THR 81 42. 571 44. 571 63. 078 1. 00 27. 38 AAAA ATOM 756 CB THR 81 41. 646 45. 222 62. 002 1. 00 28. 45 AAAA ATOM 757 OG1 THR 81 40.486 45.773 62.625 1.00 39. 19 AAAA ATOM 758 HG1 THR 81 40. 319 46. 609 62. 180 1. 00 0. 00 AAAA ATOM 759 CG2 THR 81 41. 19444. 214 60. 995 1. 00 31. 37 AAAA ATOM 760 C THR 81 41. 806 43. 489 63. 857 1. 00 27. 08 AAAA ATOM 761 O THR 81 41.840 42.302 63.503 1.00 25. 09 AAAA ATOM 762 N SER 82 41. 203 43. 873 64. 978 1. 00 26. 66 AAAA ATOM 763 H SER 82 41. 296 44. 807 65. 280 1. 00 0. 00 AAAA

ATOM 764 CA SER 82 40.425 42.912 65.764 1.00 27. 37 AAAA ATOM 765 CB SER 82 39. 992 43. 506 67. 113 1. 00 27. 54 AAAA ATOM 766 OG SER 82 39. 073 44. 565 66. 929 1. 00 32. 64 AAAA ATOM 767 HG SER 82 38. 160 44. 228 66. 813 1. 00 0. 00 AAAA ATOM 768 C SER 82 39. 192 42. 404 65. 014 1. 00 23. 10 AAAA ATOM 769 0 SER 82 38. 628 41. 385 65. 374 1. 00 24. 03 AAAA ATOM 770 N SER 83 38. 790 43. 105 63. 962 1. 00 25. 20 AAAA ATOM 771 H SER 83 39. 20143. 984 63. 811 1. 00 0. 00 AAAA ATOM 772 CA SER 83 37. 682 42. 640 63. 126 1. 00 27. 82 AAAA ATOM 773 CB SER 83 37. 339 43. 671 62. 046 1. 00 31. 00 AAAA ATOM 774 OG SER 83 37. 002 44. 929 62. 602 1. 00 44. 02 AAAA ATOM 775 HG SER 83 37. 20145. 629 61. 989 1. 00 0. 00 AAAA ATOM 776 C SER 83 38. 014 41. 315 62. 432 1. 00 27. 88 AAAA ATOM 777 0 SER 83 37. 114 40. 593 61. 985 1. 00 29. 20 AAAA ATOM 778 N VAL 84 39. 303 41. 087 62. 191 1. 00 23. 83 AAAA ATOM 779 H VAL 84 39. 969 41. 681 62. 581 1. 00 0. 00 AAAA ATOM 780 CA VAL 84 39. 723 39. 940 61. 402 1. 00 20. 73 AAAA ATOM 781 CB VAL 84 41. 231 39. 971 61. 120 1. 00 20. 85 AAAA ATOM 782 CG1 UAL 84 41. 681 38. 634 60. 533 1. 00 21. 87 AAAA ATOM 783 CG2 VAL 84 41. 536 41. 072 60. 152 1. 00 20. 91 AAAA ATOM 784 C VAL 84 39. 38338. 619 62. 064 1. 00 19. 51 AAAA ATOM 785 0 VAL 84 39. 745 38. 379 63. 206 1. 00 20. 68 AAAA ATOM 786 N TYR 85 38. 640 37. 780 61. 352 1. 00 19. 17 AAAA ATOM 787 H TYR 85 38. 292 38. 118 60. 502 1. 00 0. 00 AAAA ATOM 788 CA TYR 85 38. 314 36. 450 61. 837 1. 00 18. 54 AAAA ATOM 789 CB TYR 85 36. 921 36. 008 61. 331 1. 00 18. 00 AAAA ATOM 790 CG TYR 85 36.466 34.641 61.825 1.00 15. 07 AAAA ATOM 791 CD1 TYR 85 35. 489 34. 517 62. 828 1. 00 18. 67 AAAA ATOM 792 CE1 TYR 85 35. 074 33. 258 63. 284 1. 00 13. 74 AAAA ATOM 793 CD2 TYR 85 37. 012 33. 469 61. 299 1. 00 14. 59 AAAA ATOM 794 CE2 TYR 85 36. 619 32. 219 61. 754 1. 00 14. 12 AAAA ATOM 795 CZ TYR 85 35. 637 32. 122 62. 741 1. 00 15. 13 AAAA ATOM 796 OH TYR 85 35. 201 30. 885 63. 110 1. 00 15. 74 AAAA ATOM 797 HH TYR 85 34. 434 30. 990 63. 679 1. 00 0. 00 AAAA ATOM 798 C TYR 85 39. 388 35. 494 61. 339 1. 00 18. 88 AAAA ATOM 799 0 TYR 85 39. 503 35. 234 60. 137 1. 00 17. 70 AAAA ATOM 800 N PHE 86 40.173 34.976 62.269 1.00 17. 16 AAAA ATOM 801 H PHE 86 40. 037 35. 264 63. 201 1. 00 0. 00 AAAA ATOM 802 CA PHE 86 41. 227 34. 031 61. 938 1. 00 17. 14 AAAA ATOM 803 CB PHE 86 42. 609 34. 633 62. 294 1. 00 18. 81 AAAA ATOM 804 CG PHE 86 43. 792 33. 771 61. 884 1. 00 17. 09 AAAA

ATOM 805 CD1 PHE 86 43. 981 33. 389 60. 551 1. 00 12. 60 AAAA ATOM 806 CD2 PHE 86 44. 690 33. 306 62. 854 1. 00 16. 07 AAAA ATOM 807 CE1 PHE 86 45. 016 32. 547 60. 198 1. 00 14. 83 AAAA ATOM 808 CE2 PHE 86 45. 725 32. 467 62. 520 1. 00 14. 81 AAAA ATOM 809 CZ PHE 86 45. 892 32. 070 61. 188 1. 00 17. 97 AAAA ATOM 810 C PHE 86 41. 002 32. 701 62. 650 1. 00 15. 56 AAAA ATOM 811 0 PHE 86 40. 66432. 654 63. 837 1. 00 17. 84 AAAA ATOM 812 N MET 87 41. 296 31. 626 61. 931 1. 00 13. 32 AAAA ATOM 813 H MET 87 41. 818 31. 793 61. 129 1. 00 0. 00 AAAA ATOM 814 CA MET 87 41. 169 30. 274 62. 428 1. 00 14. 74 AAAA ATOM 815 CB MET 87 39. 993 29. 606 61. 716 1. 00 16. 52 AAAA ATOM 816 CG MET 87 39. 985 28. 110 61. 685 1. 00 22. 12 AAAA ATOM 817 SD MET 87 38. 551 27. 590 60. 704 1. 00 27. 94 AAAA ATOM 818 CE MET 87 37. 291 27. 521 61. 947 1. 00 28. 36 AAAA ATOM 819 C MET 87 42. 462 29. 523 62. 118 1. 00 18. 15 AAAA ATOM 820 0 MET 87 42. 961 29. 560 60. 983 1. 00 16. 50 AAAA ATOM 821 N LYS 88 42. 988 28. 824 63. 115 1. 00 17. 57 AAAA $ATOM 822 H LYS 88 42.582 28.893 64.004 1.00 0. 00 AAAA ATOM 823 CA LYS 88 44. 121 27. 929 62. 896 1. 00 18. 62 AAAA ATOM 824 CB LYS 88 44. 778 27. 577 64. 237 1. 00 19. 52 AAAA ATOM 825 CG LYS 88 45. 534 28. 739 64. 872 1. 00 24. 91 AAAA ATOM 826 CD LYS 88 46. 825 29. 043 64. 112 1. 00 24. 92 AAAA ATOM 827 CE LYS 88 47. 815 27. 882 64. 223 1. 00 24. 92 AAAA ATOM 828 NZ LYS 88 48. 821 27. 887 63. 118 1. 00 25. 56 AAAA ATOM 829 HZ1 LYS 88 49. 214 28. 843 63. 013 1. 00 0. 00 AAAA ATOM 830 HZ2 LYS 88 48. 349 27. 612 62. 237 1. 00 0. 00 AAAA ATOM 831 HZ3 LYS 88 49. 584 27. 213 63. 330 1. 00 0. 00 AAAA ATOM 832 C LYS 88 43. 749 26. 644 62. 146 1. 00 19. 29 AAAA ATOM 833 0 LYS 88 42. 616 26. 158 62. 220 1. 00 21. 43 AAAA ATOM 834 N GLN 89 44. 728 26. 082 61. 450 1. 00 16. 58 AAAA ATOM 835 H GLN 89 45. 585 26. 556 61. 420 1. 00 0. 00 AAAA ATOM 836 CA GLN 89 44. 599 24. 779 60. 819 1. 00 17. 18 AAAA ATOM 837 CB GLN 89 45. 271 24. 813 59. 456 1. 00 18. 38 AAAA ATOM 838 CG GLN 89 45. 350 23. 485 58. 760 1. 00 18. 24 AAAA ATOM 839 CD GLN 89 45. 853 23. 651 57. 355 1. 00 24. 51 AAAA ATOM 840 OE1 GLN 89 45. 367 24. 502 56. 594 1. 00 26. 71 AAAA ATOM 841 NE2 GLN 89 46. 881 22. 904 57. 017 1. 00 25. 25 AAAA ATOM 842 HE21 GLN 89 47. 154 22. 895 56. 079 1. 00 0. 00 AAAA ATOM 843 HE22 GLN 89 47. 349 22. 434 57. 721 1. 00 0. 00 AAAA ATOM 844 C GLN 89 45. 241 23. 691 61. 658 1. 00 20. 11 AAAA ATOM 845 0 GLN 89 46. 413 23. 781 61. 998 1. 00 22. 59 AAAA

ATOM 846 N THR 90 44. 517 22. 603 61. 869 1. 00 18. 50 AAAA ATOM 847 H. THR 90 43. 585 22. 629 61. 598 1. 00 0. 00 AAAA ATOM 848 CA THR 90 45. 072 21. 439 62. 550 1. 00 21. 73 AAAA ATOM 849 CB THR 90 44. 283 21. 115 63. 812 1. 00 24. 69 AAAA ATOM 850 OG1 THR 90 42. 924 20. 794 63. 464 1. 00 30. 17 AAAA ATOM 851 HG1 THR 90 42. 428 21. 483 62. 994 1. 00 0. 00 AAAA ATOM 852 CG2 THR 90 44. 301 22. 312 64. 752 1. 00 28. 14 AAAA ATOM 853 C THR 90 45. 102 20. 191 61. 676 1. 00 24. 47 AAAA ATOM 854 0 THR 90 45. 846 19. 252 61. 956 1. 00 28. 80 AAAA ATOM 855 N ILE 91 44. 271 20. 171 60. 632 1. 00 22. 87 AAAA ATOM 856 H ILE 91 43. 593 20. 871 60. 590 1. 00 0. 00 AAAA ATOM 857 CA ILE 91 44. 258 19. 096 59. 633 1. 00 22. 19 AAAA ATOM 858 CB ILE 91 42. 921 18. 326 59. 639 1. 00 22. 46 AAAA ATOM 859 CG2 ILE 91 42. 989 17. 157 58. 695 1. 00 18. 95 AAAA ATOM 860 CG1 ILE 91 42. 591 17. 837 61. 050 1. 00 24. 03 AAAA ATOM 861 CD ILE 91 41. 259 17. 099 61. 123 1. 00 24. 83 AAAA ATOM 862 C ILE 91 44. 422 19. 717 58. 245 1. 00 23. 19 AAAA ATOM 863 0 ILE 91 43. 626 20. 562 57. 846 1. 00 23. 87 AAAA ATOM 864 N SER 92 45. 413 19. 266 57. 487 1. 00 23. 54 AAAA ATOM 865 H SER 92 45. 791 18. 403 57. 734 1. 00 0. 00 AAAA ATOM 866 CA SER 92 45. 793 19. 943 56. 248 1. 00 23. 64 AAAA ATOM 867 CB SER 92 47. 119 19. 387 55. 737 1. 00 25. 35 AAAA ATOM 868 OG SER 92 46. 993 18. 012 55. 412 1. 00 35. 43 AAAA ATOM 869 HG SER 92 46. 79717. 917 54. 451 1. 00 0. 00 AAAA ATOM 870 C SER 92 44. 735 19. 799 55. 165 1. 00 22. 11 AAAA ATOM 871 0 SER 92 44. 459 20. 730 54. 423 1. 00 22. 66 AAAA ATOM 872 N ASN 93 44. 099 18. 644 55. 117 1. 00 21. 74 AAAA ATOM 873 H ASN 93 44. 41417. 915 55. 705 1. 00 0. 00 AAAA ATOM 874 CA ASN 93 43. 007 18. 434 54. 183 1. 00 27. 91 AAAA ATOM 875 CB ASN 93 42. 571 16. 979 54. 207 1. 00 35. 85 AAAA ATOM 876 CG ASN 93 43. 673 16. 057 53. 764 1. 00 47. 97 AAAA ATOM 877 OD1 ASN 93 44. 253 16. 233 52. 684 1. 00 52. 33 AAAA ATOM 878 ND2 ASN 93 44. 076 15. 160 54. 652 1. 00 54. 60 AAAA ATOM 879 HD21 ASN 93 43. 681 15. 126 55. 548 1. 00 0. 00 AAAA ATOM 880 HD22ASN 93 44. 780 14. 587 54. 290 1. 00 0. 00 AAAA ATOM 881 C ASN 93 41. 793 19. 323 54. 428 1. 00 25. 96 AAAA ATOM 882 0 ASN 93 40. 897 19. 373 53. 585 1. 00 24. 44 AAAA ATOM 883 N ALA 94 41. 760 19. 994 55. 583 1. 00 24. 06 AAAA ATOM 884 H ALA 94 42. 45419. 832 56. 247 1. 00 0. 00 AAAA ATOM 885 CA ALA 94 40. 690 20. 934 55. 921 1. 00 21. 31 AAAA ATOM 886 CB ALA 94 40. 516 21. 022 57. 440 1. 00 16. 74 AAAA

ATOM 887 C ALA 94 40. 940 22. 325 55. 354 1. 00 19. 83 AAAA ATOM 888 0 ALA 94 40. 089 23. 195 55. 464 1. 00 18. 59 AAAA ATOM 889 N CYS 95 42. 103 22. 544 54. 744 1. 00 17. 72 AAAA ATOM 890 H CYS 95 42. 699 21. 784 54. 585 1. 00 0. 00 AAAA ATOM 891 CA CYS 95 42. 480 23. 899 54. 322 1. 00 17. 66 AAAA ATOM 892 CB CYS 95 43. 908 23. 905 53. 752 1. 00 17. 95 AAAA ATOM 893 SG CYS 95 44. 08723. 036 52. 185 1. 00 18. 91 AAAA ATOM 894 C CYS 95 41. 503 24. 581 53. 325 1. 00 14. 55 AAAA ATOM 895 0 CYS 95 41. 304 25. 792 53. 388 1. 00 12. 57 AAAA ATOM 896 N GLY 96 40. 878 23. 808 52. 439 1. 00 16. 10 AAAA ATOM 897 H GLY 96 41. 029 22. 839 52. 444 1. 00 0. 00 AAAA ATOM 898 CA GLY 96 39. 900 24. 384 51. 525 1. 00 14. 24 AAAA ATOM 899 C GLY 96 38. 660 24. 858 52. 274 1. 00 13. 81 AAAA ATOM 900 0 GLY 96 38. 220 25. 996 52. 109 1. 00 12. 17 AAAA ATOM 901 N THR 97 38. 141 24. 009 53. 164 1. 00 13. 86 AAAA ATOM 902 H THR 97 38. 518 23. 101 53. 219 1. 00 0. 00 AAAA ATOM 903 CA THR 97 37. 033 24. 406 54. 037 1. 00 14. 26 AAAA ATOM 904 CB THR 97 36. 597 23. 257 54. 922 1. 00 14. 44 AAAA ATOM 905 OG1 THR 97 36. 020 22. 234 54. 099 1. 00 17. 53 AAAA ATOM 906 HG1 THR 97 35. 307 21. 820 54. 623 1. 00 0. 00 AAAA ATOM 907 CG2 THR 97 35. 580 23. 736 55. 981 1. 00 16. 30 AAAA ATOM 908 C THR 97 37. 383 25. 596 54. 927 1. 00 14. 53 AAAA ATOM 909 0 THR 97 36. 600 26. 539 55. 051 1. 00 13. 21 AAAA ATOM 910 N ILE 98 38. 603 25. 614 55. 455 1. 00 14. 07 AAAA ATOM 911 H ILE 98 39. 176 24. 833 55. 314 1. 00 0. 00 AAAA ATOM 912 CA ILE 98 39. 058 26. 755 56. 242 1. 00 13. 22 AAAA ATOM 913 CB ILE 98 40. 417 26. 468 56. 920 1. 00 12. 18 AAAA ATOM 914 CG2 ILE 98 40. 910 27. 704 57. 610 1. 00 11. 29 AAAA ATOM 915 CG1 ILE 98 40. 260 25. 356 57. 946 1. 00 12. 23 AAAA ATOM 916 CD ILE 98 41. 556 24. 629 58. 268 1. 00 15. 43 AAAA ATOM 917 C ILE 98 39. 156 28. 017 55. 404 1. 00 11. 49 AAAA ATOM 918 0 ILE 98 38. 755 29. 093 55. 853 1. 00 12. 87 AAAA ATOM 919 N GLY 99 39. 597 27. 885 54. 157 1. 00 11. 22 AAAA ATOM 920 H GLY 99 39. 875 27. 007 53. 832 1. 00 0. 00 AAAA ATOM 921 CA GLY 99 39. 628 29. 044 53. 281 1. 00 9. 85 AAAA ATOM 922 C GLY 99 38. 244 29. 632 53. 007 1. 00 9. 08 AAAA ATOM 923 0 GLY 99 38. 036 30. 835 53. 040 1. 00 9. 42 AAAA ATOM 924 N LEU 100 37. 277 28. 747 52. 841 1. 00 12. 12 AAAA ATOM 925 H LEU 100 37. 522 27. 794 52. 840 1. 00 0. 00 AAAA ATOM 926 CA LEU 100 35. 900 29. 148 52. 602 1. 00 15. 16 AAAA ATOM 927 CB LEU 100 35. 078 27. 891 52. 267 1. 00 18. 24 AAAA

ATOM 928 CG LEU 100 33. 723 28. 022 51. 575 1. 00 24. 26 AAAA ATOM 929 CD1 LEU 100 33. 831 29. 058 50. 465 1. 00 26. 09 AAAA ATOM 930 CD2 LEU 100 33. 289 26. 660 51. 015 1. 00 21. 41 AAAA ATOM 931 C LEU 100 35. 347 29. 870 53. 838 1. 00 13. 53 AAAA ATOM 932 0 LEU. 100 34. 837 30. 980 53. 740 1. 00 14. 20 AAAA ATOM 933 N ILE 101 35. 598 29. 300 55. 013 1. 00 13. 77 AAAA ATOM 934 H ILE 101 36. 051 28. 431 55. 000 1. 00 0. 00 AAAA ATOM 935 CA ILE 101 35. 205 29. 911 56. 281 1. 00 12. 23 AAAA ATOM 936 CB ILE 101 35. 571 28. 996 57. 462 1. 00 13. 26 AAAA ATOM 937 CG2 ILE 101 35. 402 29. 734 58. 786 1. 00 15. 98 AAAA ATOM 938 CG1 ILE 101 34. 697 27. 732 57. 405 1. 00 11. 45 AAAA ATOM 939 CD ILE 101 34. 958 26. 730 58. 500 1. 00 16. 30 AAAA ATOM 940 C ILE 101 35. 829 31. 283 56. 483 1. 00 14. 03 AAAA ATOM 941 0 ILE 101 35. 126 32. 245 56. 782 1. 00 12. 42 AAAA ATOM 942 N HIS 102 37. 120 31. 421 56. 177 1. 00 13. 04 AAAA ATOM 943 H HIS 102 37. 593 30. 615 55. 893 1. 00 0. 00 AAAA ATOM 944 CA HIS 102 37. 769 32. 738 56. 239 1. 00 10. 47 AAAA ATOM 945 CB HIS 102 39. 265 32. 613 55. 897 1. 00 13. 05 AAAA ATOM 946 CG HIS 102 40.107 32.125 57.030 1.00 10. 70 AAAA ATOM 947 CD2 HIS 102 40. 153 32. 499 58. 332 1. 00 13. 91 AAAA ATOM 948 ND1 HIS 102 41. 088 31. 168 56. 877 1. 00 11. 81 AAAA ATOM 949 HD1 HIS 102 41. 295 30. 682 56. 041 1. 00 0. 00 AAAA ATOM 950 CE1 HIS 102 41. 708 30. 974 58. 028 1. 00 11. 88 AAAA ATOM 951 NE2 HIS 102 41. 162 31. 778 58. 927 1. 00 15. 13 AAAA ATOM 952 HE2 HIS 102 41. 468 31. 860 59. 843 1. 00 0. 00 AAAA ATOM 953 C HIS 102 37. 125 33. 757 55. 291 1. 00 11. 19 AAAA ATOM 954 0 HIS 102 36. 969 34. 935 55. 640 1. 00 12. 51 AAAA ATOM 955 N ALA 103 36. 844 33. 328 54. 055 1. 00 13. 10 AAAA ATOM 956 H ALA 103 37. 020 32. 394 53. 814 1. 00 0. 00 AAAA ATOM 957 CA ALA 103 36. 264 34. 227 53. 056 1. 00 10. 96 AAAA ATOM 958 CB ALA 103 36. 140 33. 504 51. 703 1. 00 9. 09 AAAA ATOM 959 C ALA 103 34. 881 34. 712 53. 531 1. 00 15. 64 AAAA ATOM 960 0 ALA 103 34. 558 35. 899 53. 440 1. 00 18. 87 AAAA ATOM 961 N ILE 104 34. 073 33. 787 54. 047 1. 00 15. 87 AAAA ATOM 962 H ILE 104 34. 402 32. 864 54. 115 1. 00 0. 00 AAAA ATOM 963 CA ILE 104 32. 701 34. 118 54. 460 1. 00 15. 42 AAAA ATOM 964 CB ILE 104 31. 813 32. 829 54. 537 1. 00 13. 84 AAAA ATOM 965 CG2 ILE 104 30. 365 33. 200 54. 915 1. 00 18. 30 AAAA ATOM 966 CG1 ILE 104 31. 822 32. 081 53. 204 1. 00 13. 16 AAAA ATOM 967 CD ILE 104 31. 510 32. 966 51. 997 1. 00 14. 57 AAAA ATOM 968 C ILE 104 32. 677 34. 862 55. 822 1. 00 15. 78 AAAA

ATOM 9690) LE10432. 05135. 92255. 9421. 0016. 92 AAAA ATOM 970 N ALA 105 33. 411 34. 345 56. 813 1. 00 16. 50 AAAA ATOM 971 H ALA 105 33. 832 33. 486 56. 631 1. 00 0. 00 AAAA ATOM 972 CA ALA 105 33. 529 34. 998 58. 121 1. 00 17. 00 AAAA ATOM 973 CB ALA 105 34. 492 34. 239 59. 022 1. 00 16. 84 AAAA ATOM 974 C ALA 105 33.963 36.448 58.039 1.00 20. 96 AAAA ATOM 975 0 ALA 105 33. 452 37. 298 58. 776 1. 00 23. 63 AAAA ATOM 976 N ASN 106 34.869 36.760 57.117 1.00 16. 81 AAAA ATOM 977 H ASN 106 35. 225 36. 046 56. 549 1. 00 0. 00 AAAA ATOM 978 CA ASN 106 35. 347 38. 132 57. 002 1. 00 17. 98 AAAA ATOM 979 CB ASN 106 36. 825 38. 128 56. 611 1. 00 17. 74 AAAA ATOM 980 CG ASN 106 37. 707 37. 690 57. 755 1. 00 14. 08 AAAA ATOM 981 OD1 ASN 106 37. 977 38. 463 58. 655 1. 00 17. 07 AAAA ATOM 982 ND2 ASN 106 38. 042 36. 415 57. 795 1. 00 14. 28 AAAA ATOM 983 HD21 ASN 106 37. 713 35. 831 57. 096 1. 00 0. 00 AAAA ATOM 984 HD22ASN 106 38. 61136. 181 58. 571 1. 00 0. 00 AAAA ATOM 985 C ASN 106 34. 522 39. 008 56. 048 1. 00 19. 28 AAAA ATOM 986 0 ASN 106 34. 898 40. 129 55. 748 1. 00 17. 22 AAAA ATOM 987 N ASN 107 33. 389 38. 478 55. 584 1. 00 21. 43 AAAA ATOM 988 H ASN 107 33. 219 37. 539 55. 782 1. 00 0. 00 AAAA ATOM 989 CA ASN 107 32. 445 39. 213 54. 739 1. 00 21. 54 AAAA ATOM 990 CB ASN 107 32. 611 38. 786 53. 296 1. 00 18. 81 AAAA ATOM 991 CG ASN 107 33. 897 39. 275 52. 710 1. 00 21. 63 AAAA ATOM 992 OD1 ASN 107 34. 079 40. 472 52. 495 1. 00 22. 45 AAAA ATOM 993 ND2 ASN 107 34. 833 38. 368 52. 514 1. 00 17. 35 AAAA ATOM 994 HD21 ASN 107 34. 605 37. 441 52. 622 1. 00 0. 00 AAAA ATOM 995 HD22ASN 107 35. 709 38. 734 52. 238 1. 00 0. 00 AAAA ATOM 996 C ASN 107 31. 006 38. 927 55. 177 1. 00 25. 95 AAAA ATOM 997 O ASN 107 30.073 39.091 54.399 1.00 26. 08 AAAA ATOM 998 N LYS 108 30.856 38.514 56.435 1.00 27. 04 AAAA ATOM 999 H LYS 108 31. 660 38. 510 56. 994 1. 00 0. 00 AAAA ATOM 1000 CA LYS 108 29. 611 37. 983 56. 984 1. 00 30. 55 AAAA ATOM 1001 CB LYS 108 29. 821 37. 766 58. 483 1. 00 31. 98 AAAA ATOM 1002 CG LYS 108 28. 880 36. 783 59. 136 1. 00 39. 13 AAAA ATOM 1003 CD LYS 108 29. 42036. 324 60. 493 1. 00 44. 06 AAAA ATOM 1004 CE LYS 108 29. 708 37. 491 61. 442 1. 00 45. 91 AAAA ATOM 1005 NZ LYS 108 29. 833 37. 023 62. 849 1. 00 46. 37 AAAA ATOM 1006 HZ1 LYS 108 30. 640 36. 383 62. 912 1. 00 0. 00 AAAA ATOM 1007 HZ2 LYS 108 28. 963 36. 525 63. 134 1. 00 0. 00 AAAA ATOM 1008 HZ3 LYS 108 29. 992 37. 838 63. 478 1. 00 0. 00 AAAA ATOM 1009 C LYS 108 28. 400 38. 907 56. 732 1. 00 30. 21 AAAA

ATOM 1010 0 LYS 108 27. 330 38. 462 56. 309 1. 00 28. 80 AAAA ATOM 1011 N ASP 109 28. 647 40. 206 56. 792 1. 00 31. 65 AAAA ATOM 1012HASP109 29. 573 40. 464 56. 950 1. 00 0. 00 AAAA ATOM 1013 CA ASP 109 27. 601 41. 191 56. 575 1. 00 34. 09 AAAA ATOM 1014 CB ASP 109 27. 982 42. 501 57. 274 1. 00 38. 39 AAAA ATOM 1015 CG ASP 109 28. 062 42. 354 58. 800 1. 00 43. 78 AAAA ATOM 1016001ASP109 28. 880 43. 070 59. 420 1. 00 45. 93 AAAA ATOM 1017 OD2 ASP 109 27. 300 41. 549 59. 384 1. 00 47. 42 AAAA ATOM 1018 C ASP 109 27. 247 41.456 55.109 1.00 33. 23 AAAA ATOM 1019 0 ASP 109 26. 350 42.237 54.827 1.00 37. 51 AAAA ATOM 1020 N LYS 110 27. 966 40. 836 54. 176 1. 00 30. 81 AAAA ATOM 1021 H LYS 110 28. 78140. 371 54. 456 1. 00 0. 00 AAAA ATOM 1022 CA LYS 110 27. 619 40. 913 52. 747 1. 00 26. 81 AAAA ATOM 1023 CB LYS 110 28. 877 40. 922 51. 873 1. 00 26. 19 AAAA ATOM 1024 CG LYS 110 29. 898 41.966 52.267 1.00 31. 88 AAAA ATOM 1025 CD LYS 110 31. 034 42. 039 51. 252 1. 00 35. 86 AAAA ATOM 1026 CE LYS 110 32. 009 43. 165 51. 577 1. 00 35. 07 AAAA ATOM 1027 NZ LYS 110 32. 734 42. 922 52. 858 1. 00 40. 57 AAAA ATOM 1028 HZ1 LYS 110 33. 276 42. 042 52. 794 1. 00 0. 00 AAAA ATOM 1029 HZ2 LYS 110 32. 047 42. 849 53. 637 1. 00 0. 00 AAAA ATOM 1030 HZ3 LYS 110 33. 388 43. 708 53. 047 1. 00 0. 00 AAAA ATOM 1031 C LYS 110 26. 772 39. 722 52. 335 1. 00 23. 39 AAAA ATOM 1032 0 LYS 110 26. 329 39. 623 51. 190 1. 00 23. 47 AAAA ATOM 1033 N MET 111 26. 72738. 729 53. 210 1. 00 24. 31 AAAA ATOM 1034 H MET 111 27. 069 38. 886 54. 106 1. 00 0. 00 AAAA ATOM 1035 CA MET 111 26. 133 37. 439 52. 884 1. 00 24. 62 AAAA ATOM 1036 CB MET 111 26. 875 36. 314 53. 595 1. 00 25. 74 AAAA ATOM 1037 CG MET 111 28. 355 36. 281 53. 307 1. 00 27. 29 AAAA ATOM 1038 SO MET 111 28. 669 35. 921 51. 605 1. 00 28. 33 AAAA ATOM 1039 CE MET 111 29. 842 37. 263 51. 197 1. 00 22. 52 AAAA ATOM 1040 C MET 111 24. 684 37. 388 53. 313 1. 00 25. 26 AAAA ATOM 1041 0 MET 111 24. 304 37.974 54.325 1.00 24. 63 AAAA ATOM 1042 N HIS 112 23. 947 36. 513 52. 656 1. 00 25. 79 AAAA ATOM 1043 H HIS 112 24. 366 36. 111 51. 894 1. 00 0. 00 AAAA ATOM 1044 CA HIS 112 22. 548 36. 291 52. 965 1. 00 27. 47 AAAA ATOM 1045 CB HIS 112 21. 683 36.549 51.723 1.00 35. 41 AAAA ATOM 1046 CG HIS 112 20. 209 36.375 51.954 1.00 42. 70 AAAA ATOM 1047 CD2 HIS 112 19. 494 36.271 53.103 1.00 43. 97 AAAA ATOM 1048 ND1 HIS 112 19. 300 36.261 50.924 1.00 44. 16 AAAA ATOM 1049 HD1 HIS 112 19. 465 36. 489 49. 979 1. 00 0. 00 AAAA ATOM 1050 CE1 HIS 112 18. 090 36. 100 51. 426 1. 00 46. 54 AAAA

ATOM 1051 NE2 HIS 112 18. 180 36. 091 52. 742 1. 00 46. 27 AAAA ATOM 1052 HE2 HIS 112 17. 442 36. 197 53. 384 1. 00 0. 00 AAAA ATOM 1053 C HIS 112 22. 391 34. 867 53. 431 1. 00 24. 85 AAAA ATOM 1054 0 HIS 112 22. 325 33. 931 52. 618 1. 00 23. 17 AAAA ATOM 1055 N PHE 113 22. 357 34. 714 54. 748 1. 00 24. 82 AAAA ATOM 1056 H PHE 113 22. 542 35. 493 55. 300 1. 00 0. 00 AAAA ATOM 1057 CA PHE 113 22. 154 33. 414 55. 374 1. 00 29. 11 AAAA ATOM 1058 CB PHE 113 22. 758 33. 401 56. 794 1. 00 27. 84 AAAA ATOM 1059 CG PHE 113 24. 247 33. 637 56. 832 1. 00 24. 66 AAAA ATOM 1060 CD1 PHE 113 24. 753 34. 910 57. 086 1. 00 23. 03 AAAA ATOM 1061 CD2 PHE 113 25. 141 32. 586 56. 598 1. 00 25. 31 AAAA ATOM 1062 CE1 PHE 113 26. 133 35. 146 57. 096 1. 00 27. 19 AAAA ATOM 1063 CE2 PHE 113 26. 520 32. 814 56. 608 1. 00 24. 97 AAAA ATOM 1064 CZ PHE 113 27. 014 34. 100 56. 858 1. 00 24. 09 AAAA ATOM 1065 C PHE 113 20. 670 33. 008 55. 449 1. 00 31. 00 AAAA ATOM 1066 0 PHE 113 19. 790 33. 848 55. 686 1. 00 33. 09 AAAA ATOM 1067 N GLU 114 20. 402 31. 772 55. 043 1. 00 29. 35 AAAA ATOM 1068 H GLU 114 21. 11131. 342 54. 547 1. 00 0. 00 AAAA ATOM 1069 CA GLU 114 19. 173 31. 085 55. 391 1. 00 29. 30 AAAA ATOM 1070 CB GLU 114 19. 167 29. 712 54. 748 1. 00 30. 12 AAAA ATOM 1071 CG GLU 114 19. 228 29. 750 53. 232 1. 00 32. 89 AAAA ATOM 1072 CD GLU 114 19. 340 28. 379 52. 624 1. 00 34. 82 AAAA ATOM 1073 OE1 GLU 114 19. 097 27. 384 53. 338 1. 00 37. 36 AAAA ATOM 1074 OE2 GLU 114 19. 713 28. 293 51. 437 1. 00 43. 01 AAAA ATOM 1075 C GLU 114 19. 067 30. 927 56. 903 1. 00 32. 03 AAAA ATOM 1076 0 GLU 114 20. 080 30. 731 57. 575 1. 00 30. 35 AAAA ATOM 1077 N SER 115 17.836 30.830 57.403 1.00 32. 06 AAAA ATOM 1078 H SER 115 17. 081 30. 845 56. 781-1. 00 0. 00 AAAA ATOM 1079 CA SER 115 17. 596 30. 797 58. 849 1. 00 34. 83 AAAA ATOM 1080 CB SER 115 16. 098 30. 682 59. 157 1. 00 37. 46 AAAA ATOM 1081 OG SER 115 15. 337 31. 576 58. 368 1. 00 40. 20 AAAA ATOM 1082 HG SER 115 15. 254 31. 197 57. 494 1. 00 0. 00 AAAA ATOM 1083 C SER 115 18. 324 29. 642 59. 522 1. 00 32. 68 AAAA ATOM 1084 0 SER 115 18. 964 29. 820 60. 551 1. 00 34. 71 AAAA ATOM 1085 N GLY 116 18. 230 28. 461 58. 931 1. 00 28. 08 AAAA ATOM 1086 H GLY 116 17. 723 28. 386 58. 104 1. 00 0. 00 AAAA ATOM 1087 CA GLY 116 18. 890 27. 314 59. 525 l. OO 30. 75 AAAA ATOM 1088 C GLY 116 20. 331 27. 077 59. 087 1. 00 29. 04 AAAA ATOM 1089 0 GLY 116 20. 821 25. 954 59. 221 1. 00 27. 89 AAAA ATOM 1090 N SER 117 21. 011 28. 123 58. 601 1. 00 28. 86 AAAA ATOM 1091 H SER 117 20.613 29.011 58.704 1.00 0. 00 AAAA

ATOM 1092 CA SER 117 22. 365 28. 000 58. 017 1. 00 25. 44 AAAA ATOM 1093 CB SER 117 22. 867 29. 378 57. 561 1. 00 23. 69 AAAA ATOM 1094 OG SER 117 24. 260 29. 369 57. 281 1. 00 21. 52 AAAA ATOM 1095 HG SER 117 24. 415 28. 790 56. 527 1. 00 0. 00 AAAA ATOM 1096 C SER 117 23. 35227. 412 59. 027 1. 00 24. 07 AAAA ATOM 1097 0 SER 117 23. 536 27. 980 60. 107 1. 00 23. 98 AAAA ATOM 1098 N THR 118 23. 959 26. 271 58. 701 1. 00 22. 14 AAAA ATOM 1099 H THR 118 23. 71125. 878 57. 836 1. 00 0. 00 AAAA ATOM 1100 CA THR 118 24. 950 25. 660 59. 593 1. 00 23. 25 AAAA ATOM 1101 CB THR 118 25. 389 24. 266 59. 128 1. 00 26. 09 AAAA ATOM 1102 OG1 THR 118 25. 837 24. 326 57. 771 1. 00 26. 52 AAAA ATOM 1103 HG1 THR 118 25. 089 24. 335 57. 150 1. 00 0. 00 AAAA ATOM 1104 CG2 THR 118 24. 255 23. 298 59. 235 1. 00 28. 30 AAAA ATOM 1105 C THR 118 26. 219 26. 506 59. 721 1. 00 25. 21 AAAA ATOM 1106 0 THR 118 26. 783 26. 627 60. 806 1. 00 23. 40 AAAA ATOM 1107 N LEU 119 26. 662 27. 117 58. 623 1. 00 24. 77 AAAA ATOM 1108 H LEU 119 26. 247 26. 892 57. 763 1. 00 0. 00 AAAA ATOM 1109 CA LEU 119 27. 793 28. 025 58. 711 1. 00 21. 55 AAAA ATOM 1110 CB LEU 119 28. 278 28. 446 57. 317 1. 00 21. 35 AAAA ATOM 1111 CG LEU 119 29. 527 29. 346 57. 292 1. 00 22. 12 AAAA ATOM 1112 CD1 LEU 119 30. 675 28. 670 58. 031 1. 00 21. 90 AAAA ATOM 1113 CD2 LEU 119 29. 938 29. 618 55. 852 1. 00 25. 56 AAAA ATOM 1114 C LEU 119 27. 447 29. 257 59. 547 1. 00 22. 56 AAAA ATOM 11150LEU11928. 24929. 68060. 3771. 0021. 20 AAAA ATOM 1116 N LYS 120 26. 240 29. 800 59. 384 1. 00 19. 77 AAAA ATOM 1117 H LYS 120 25. 624 29. 429 58. 719 1. 00 0. 00 AAAA ATOM 1118 CA LYS 120 25. 847 30. 974 60. 164 1. 00 20. 58 AAAA ATOM 1119 CB LYS 120 24. 408 31. 368 59. 844 1. 00 27. 26 AAAA ATOM 1120 CG LYS 120 23. 917 32. 629 60. 548 1. 00 31. 17 AAAA ATOM 1121 CD LYS 120 22. 39832. 703 60. 494 1. 00 37. 84 AAAA ATOM 1122 CE LYS 120 21. 870 33. 951 61. 161 1. 00 41. 70 AAAA ATOM 1123 NZ LYS 120 22. 115 33. 910 62. 630 1. 00 46. 53 AAAA ATOM 1124 HZ1 LYS 120 23. 142 33. 971 62. 791 1. 00 0. 00 AAAA ATOM 1125 HZ2 LYS 120 21. 749 33. 022 63. 030 1. 00 0. 00 AAAA ATOM 1126 HZ3 LYS 120 21. 652 34. 725 63. 081 1. 00 0. 00 AAAA ATOM 1127 C LYS 120 25. 978 30. 687 61. 659 1. 00 21. 21 AAAA ATOM 1128 0 LYS 120 26. 554 31. 485 62. 406 1. 00 21. 15 AAAA ATOM 1129 N LYS 121 25. 509 29. 511 62. 066 1. 00 21. 91 AAAA ATOM 1130 H LYS 121 25. 104 28. 933 61. 385 1. 00 0. 00 AAAA ATOM 1131 CA LYS 121 25. 564 29. 109 63. 460 1. 00 22. 16 AAAA ATOM 1132 CB LYS 121 24. 764 27. 828 63. 710 1. 00 22. 21 AAAA

ATOM 1133 CG LYS 121 24. 645 27. 532 65. 204 1. 00 26. 49 AAAA ATOM 1134 CD LYS 121 24. 221 26. 114 65. 506 1. 00 30. 08 AAAA ATOM 1135 CE LYS 121 24. 099 25. 911 67. 019 1. 00 32. 98 AAAA ATOM 1136 NZ LYS 121 23. 937 24. 481 67. 402 1. 00 34. 13 AAAA ATOM 1137 HZ1 LYS 121 24. 516 23. 891 66. 772 1. 00 0. 00 AAAA ATOM 1138 HZ2 LYS 121 22. 943 24. 188 67. 309 1. 00 0. 00 AAAA ATOM 1139 HZ3 LYS 121 24. 240 24. 348 68. 384 1. 00 0. 00 AAAA ATOM 1140 C LYS 121 26. 994 28. 898 63. 946 1. 00 23. 88 AAAA ATOM 1141 0 LYS 121 27. 315 29. 236 65. 088 1. 00 24. 24 AAAA ATOM 1142 N PHE 122 27. 834 28. 301 63. 104 1. 00 21. 06 AAAA ATOM 1143 H PHE 122 27. 476 27. 916 62. 273 1. 00 0. 00 AAAA ATOM 1144 CA PHE 122 29. 258 28. 160 63. 437 1. 00 20. 87 AAAA ATOM 1145 CB PHE 122 30. 001 27. 412 62. 333 1. 00 18. 89 AAAA ATOM 1146 CG PHE 122 31. 468 27. 264 62. 599 1. 00 21. 64 AAAA ATOM 1147 CD1 PHE 122 31. 950 26. 174 63. 324 1. 00 21. 50 AAAA ATOM 1148 CD2 PHE 122 32. 369 28. 255 62. 192 1. 00 21. 09 AAAA ATOM 1149 CE1 PHE 122 33. 314 26. 065 63. 644 1. 00 18. 48 AAAA ATOM 1150 CE2 PHE 122 33. 727 28. 158 62. 515 1. 00 19. 92 AAAA ATOM 1151 CZ PHE 122 34. 192 27. 058 63. 239 1. 00 18. 04 AAAA ATOM 1152 C PHE 122 29. 934 29. 514 63. 670 1. 00 20. 43 AAAA ATOM 1153 0 PHE 122 30. 666 29. 699 64. 647 1. 00 22. 71 AAAA ATOM 1154 N LEU 123 29. 646 30. 471 62. 800 1. 00 16. 51 AAAA ATOM 1155 H LEU 123 29. 032 30. 251 62. 069 1. 00 0. 00 AAAA ATOM 1156 CA LEU 123 30. 224 31. 793 62. 905 1. 00 19. 43 AAAA ATOM 1157 CB LEU 123 29. 913 32. 592 61. 645 1. 00 16. 02 AAAA ATOM 1158 CG LEU 123 30. 559 32. 020 60. 383 1. 00 15. 88 AAAA ATOM 1159 CD1 LEU 123 30. 139 32. 849 59. 185 1. 00 16. 91 AAAA ATOM 1160 CD2 LEU 123 32. 097 32. 005 60. 562 1. 00 17. 98 AAAA ATOM 1161 C LEU 123 29. 777 32. 574 64. 137 1. 00 23. 21 AAAA ATOM 1162 0 LEU 123 30. 58133. 249 64. 777 1. 00 22. 18 AAAA ATOM 1163 N GLU 124 28. 492 32. 499 64. 469 1. 00 24. 39 AAAA ATOM 1164 H GLU 124 27. 889 31. 989 63. 882 1. 00 0. 00 AAAA ATOM 1165 CA GLU 124 28. 003 33. 237 65. 628 1. 00 25. 74 AAAA ATOM 1166 CB GLU 124 26. 474 33. 412 65. 587 1. 00 29. 50 AAAA ATOM 1167 CG GLU 124 25. 678 32. 166 65. 281 1. 00 36. 33 AAAA ATOM 1168 CD GLU 124 24. 262 32. 483 64. 795 1. 00 42. 27 AAAA ATOM 1169 OE1 GLU 124 23. 941 33. 685 64. 630 1. 00 43. 67 AAAA ATOM 1170 OE2 GLU 124 23. 474 31. 531 64. 578 1. 00 42. 51 AAAA ATOM 1171 C GLU 124 28. 439 32. 576 66. 924 1. 00 21. 63. AAAA ATOM 1172 0 GLU 124 28. 850 33. 260 67. 850 1. 00 25. 47 AAAA ATOM 1173 N GLU 125 28. 531 31. 256 66. 928 1. 00 20. 31 AAAA

ATOM 1174 H GLU 125 28. 213 30. 752 66. 151 1. 00 0. 00 AAAA ATOM 1175 CA GLU 125 29. 072 30. 570 68. 091 1. 00 24. 49 AAAA ATOM 1176 CB GLU 125 28. 836 29. 061 68. 010 1. 00 29. 56 AAAA ATOM 1177 CG GLU 125 27. 352 28. 625 68. 085 1. 00 35. 07 AAAA ATOM 1178 CD GLU 125 26. 599 29. 184 69. 303 1. 00 40. 25 AAAA ATOM 1179 OE1 GLU 125 27. 239 29. 516 70. 334 1. 00 38. 36 AAAA ATOM 1180 OE2 GLU 125 25. 353 29. 280 69. 224 1. 00 40. 90 AAAA ATOM 1181 C GLU 125 30. 558 30. 832 68. 290 1. 00 25. 02 AAAA ATOM 1182 0 GLU 125 31. 037 30. 829 69. 426 1. 00 27. 15 AAAA ATOM 1183 N SER 126 31. 291 31. 034 67. 194 1. 00 24. 83 AAAA ATOM 1184 H SER 126 30. 820 31. 102 66. 338 1. 00 0. 00 AAAA ATOM 1185 CA SER 126 32. 759 31. 135 67. 256 1. 00 22. 04 AAAA ATOM 1186 CB SER 126 33. 40630. 320 66. 130 1. 00 19. 89 AAAA ATOM 1187 OG SER 126 33. 126 30. 932 64. 880 1. 00 18. 20 AAAA ATOM 1188 HG SER 126 32. 193 30. 782 64. 685 1. 00 0. 00 AAAA ATOM 1189 C SER 126 33. 294 32. 562 67. 200 1. 00 23. 67 AAAA ATOM 1190 O SER 126 34. 50732. 768 67. 080 1. 00 25. 05 AAAA ATOM 1191 N VAL 127 32. 422 33. 533 67. 456 1. 00 22. 04 AAAA ATOM 1192 H VAL 127 31. 518 33. 298 67. 748 1. 00 0. 00 AAAA ATOM 1193 CA VAL 127 32. 757 34. 942 67. 278 1. 00 24. 22 AAAA ATOM 1194 CB VAL 127 31. 479 35. 822 67. 390 1. 00 25. 46 AAAA ATOM 1195 CG1 VAL 127 31. 117 36. 056 68. 842 1. 00 27. 14 AAAA ATOM 1196 CG2 VAL 127 31. 667 37. 136 66. 669 1. 00 29. 08 AAAA ATOM 1197 C VAL 127 33. 817 35. 436 68. 274 1. 00 25. 27 AAAA ATOM 1198 0 VAL 127 34. 596 36. 339 67. 977 1. 00 26. 45 AAAA ATOM 1199 N SER 128 33. 872 34. 807 69. 440 1. 00 25. 75 AAAA ATOM 1200 H SER 128 33. 200 34. 110 69. 594 1. 00 0. 00 AAAA ATOM 1201 CA SER 128 34. 781 35. 242 70. 497 1. 00 26. 71 AAAA ATOM 1202 CB SER 128 33. 983 35. 692 71. 723 1. 00 26. 97 AAAA ATOM 1203 OG SER 128 33. 345 36. 937 71. 487 1. 00 32. 77 AAAA ATOM 1204 HG SER 128 33. 932 37. 594 71. 090 1. 00 0. 00 AAAA ATOM 1205 C SER 128 35. 78634. 16670. 9091. 0027. 80 AAAA ATOM 1206 0 SER 128 36. 560 34. 351 71. 857 1. 00 29. 12 AAAA ATOM 1207 N MET 129 35. 807 33. 056 70. 180 1. 00 22. 86 AAAA ATOM 1208 H MET 129 35. 182 33. 002 69. 438 1. 00 0. 00 AAAA ATOM 1209 CA MET 129 36. 810 32. 030 70. 416 1. 00 20. 85 AAAA ATOM 1210 CB MET 129 36. 426 30. 738 69. 710 1. 00 21. 01 AAAA ATOM 1211 CG MET 129 35. 10930. 156 70. 115 1. 00 21. 58 AAAA ATOM 1212 SD MET 129 34. 770 28. 650 69. 190 1. 00 24. 74 AAAA ATOM 1213 CE MET 129 33. 164 28. 188 69. 880 1. 00 20. 30 AAAA ATOM 1214 C MET 129 38. 166 32. 500 69. 875 1. 00 21. 66 AAAA

ATOM 1215 0 MET 129 38. 238 33. 329 68. 961 1. 00 19. 22 AAAA ATOM 1216 N SER 130 39. 23231. 88070. 3611. 0020. 89 AAAA ATOM 1217 H SER 130 39. 092 31. 193 71. 016 1. 00 0. 00 AAAA ATOM 1218 CA SER 130 40. 568 32. 109 69. 800 1. 00 21. 92 AAAA ATOM 1219 CB SER 130 41. 633 31. 625 70. 783 1. 00 13002 AAAA ATOM 1220 OG SER 130 41. 639 30. 219 70. 811 1. 00 14. 33 AAAA ATOM 1221 HG SER 130 42. 205 29. 949 71. 540 1. 00 0. 00 AAAA ATOM 1222 C SER 130 40. 726 31. 351 68. 464 1. 00 20. 98 AAAA ATOM 1223 0 SER 130 40. 168 30. 269 68. 293 1. 00 22. 57 AAAA ATOM 1224 N PRO 131 41. 686 31. 774 67. 624 1. 00 21. 58 AAAA ATOM 1225 CD PRO 131 42. 485 33. 009 67. 725 1. 00 18. 87 AAAA ATOM 1226 CA PRO 131 42. 056 30. 997 66. 431 1. 00 19. 82 AAAA ATOM 1227 CB PRO 131 43. 341 31. 675 65. 975 1. 00 18. 16 AAAA ATOM 1228 CG PRO 131 43. 131 33. 097 66. 364 1. 00 18. 91 AAAA ATOM 1229 C PRO 131 42. 256 29. 503 66. 685 1. 00 18. 54 AAAA ATOM 1230 0 PRO 131 41. 773 28. 660 65. 939 1. 00 19. 61 AAAA ATOM 1231 N GLU 132 42. 866 29. 180 67. 811 1. 00 17. 68 AAAA ATOM 1232 H GLU 132 43. 096 29. 935 68. 378 1. 00 0. 00 AAAA ATOM 1233 CA GLU 132 43. 061 27. 789 68. 210 1. 00 20. 89 AAAA ATOM 1234 CB GLU 132 44. 060 27. 717 69. 377 1. 00 21. 09 AAAA ATOM 1235 CG GLU 132 45. 413 28. 416 69. 127 1. 00 32. 04 AAAA ATOM 1236 CD GLU 132 45. 333 29. 949 69. 017 1. 00 36. 37 AAAA ATOM 1237 OE1 GLU 132 44. 489 30. 566 69. 701 1. 00 37. 91 AAAA ATOM 1238 OE2 GLU 132 46. 148 30. 546 68. 269 1. 00 42. 82 AAAA ATOM 1239 C GLU 132 41. 727 27. 114 68. 620 1. 00 17. 56 AAAA ATOM 1240 0 GLU 132 41. 441 25. 973 68. 252 1. 00 18. 36 AAAA ATOM 1241 N GLU 133 40. 913 27. 830 69. 380 1. 00 20. 52 AAAA ATOM 1242 H GLU 133 41. 184 28. 735 69. 645 1. 00 0. 00 AAAA ATOM 1243 CA GLU 133 39. 625 27. 290 69. 796 1. 00 20. 73 AAAA ATOM 1244 CB GLU 133 38. 943 28. 259 70. 762 1. 00 23. 60 AAAA ATOM 1245 CG GLU 133 39. 479 28. 128 72. 198 1. 00 24. 26 AAAA ATOM 1246 CD GLU 133 39. 027 29. 241 73. 118 1. 00 25. 00 AAAA ATOM 1247 OE1 GLU 133 38. 859 30. 389 72. 659 1. 00 25. 09 AAAA ATOM 1248 OE2 GLU 133 38. 907 28. 977 74. 331 1. 00 28. 62 AAAA ATOM 1249 C GLU 133 38. 730 27. 019 68. 597 1. 00 18. 69 AAAA ATOM 1250 0 GLU 133 38. 093 25. 967 68. 514 1. 00 19. 64 AAAA ATOM 1251 N ARG 134 38. 800 27. 908 67. 612 1. 00 20. 05 AAAA ATOM 1252 H ARG 134 39. 396 28. 673 67. 722 1. 00 0. 00 AAAA ATOM 1253 CA ARG 134 37. 986 27. 786 66. 391 1. 00 18. 25 AAAA ATOM 1254 CB ARG 134 38. 135 29. 038 65. 543 1. 00 13. 07 AAAA ATOM 1255 CG ARG 134 37. 576 30. 266 66. 200 1. 00 12. 02 AAAA

ATOM 1256 CD ARG 134 37. 921 31. 498 65. 435 1. 00 14. 57 AAAA ATOM 1257 NE ARG 134 37. 128 32. 629 65. 891 1. 00 14. 70 AAAA ATOM 1258 HE ARG 134 36. 25132. 439 66. 282 1. 00 0. 00 AAAA ATOM 1259 CZ ARG 134 37. 518 33. 894 65. 804 1. 00 17. 08 AAAA ATOM 1260 NH1 ARG 134 36. 702 34. 867 66. 202 1. 00 19. 69 AAAA ATOM 1261 HHll ARG 134 37. 003 35. 819 66. 165 1. 00 0. 00 AAAA ATOM 1262 HH12ARG 134 35. 79134. 643 66. 549 1. 00 0. 00 AAAA ATOM 1263 NH2 ARG 134 38. 697 34. 191 65. 267 1. 00 17. 97 AAAA ATOM 1264 HH21 ARG 134 39. 284 33. 466 64. 908 1. 00 0. 00 AAAA ATOM 1265 HH22ARG 134 38. 996 35. 145 65. 226 1. 00 0. 00 AAAA ATOM 1266 C ARG 134 38. 311 26. 543 65. 566 1. 00 17. 76 AAAA ATOM 1267 0 ARG 134 37. 406 25. 854 65. 078 1. 00 17. 79 AAAA ATOM 1268 N ALA 135 39. 585 26. 156 65. 568 1. 00 16. 35 AAAA ATOM 1269 H ALA 135 40. 235 26. 734 66. 017 1. 00 0. 00 AAAA ATOM 1270 CA ALA 135 40. 009 24. 934 64. 905 1. 00 16. 21 AAAA ATOM 1271 CB ALA 135 41. 538 24. 867 64. 833 1. 00 15. 84 AAAA ATOM 1272 C ALA 135 39. 462 23. 712 65. 635 1. 00 19. 65 AAAA ATOM 1273 0 ALA 135 39. 029 22. 744 65. 010 1. 00 20. 84 AAAA ATOM 1274 N ARG 136 39. 476 23. 762 66. 963 1. 00 21. 92 AAAA ATOM 1275 H ARG 136 39. 873 24. 554 67. 393 1. 00 0. 00 AAAA ATOM 1276 CA ARG 136 38. 935 22. 677 67. 785 1. 00 22. 78 AAAA ATOM 1277 CB ARG 136 39. 268 22. 948 69. 255 1. 00 22. 88 AAAA ATOM 1278 CG ARG 136 39. 719 21. 737 70. 028 1. 00 30. 16 AAAA ATOM 1279 CD ARG 136 40. 153 22. 137 71. 432 0. 00 26. 92 AAAA ATOM 1280 NE ARG 136 40. 848 21. 059 72. 132 0. 00 27. 01 AAAA ATOM 1281 HE ARG 136 41. 821 20. 997 72. 030 0. 00 0. 00 AAAA ATOM 1282 CZ ARG 136 40. 251 20. 159 72. 908 0. 00 26. 44 AAAA ATOM 1283 NH1 ARG 136 40. 975 19. 250 73. 546 0. 00 26. 25 AAAA ATOM 1284 HH11ARG 136 41. 972 19. 258 73. 462 1. 00 0. 00 AAAA ATOM 1285 HH12ARG 136 40. 527 18. 592 74. 150 1. 00 0. 00 AAAA ATOM 1286 NH2 ARG 136 38. 930 20. 142 73. 022 0. 00 26. 23 AAAA ATOM 1287 HH21 ARG 136 38. 372 20. 799 72. 513 1. 00 0. 00 AAAA ATOM 1288 HH22ARG 136 38. 48819. 468 73. 613 1. 00 0. 00 AAAA ATOM 1289 C ARG 136 37. 400 22. 548 67. 608 1. 00 20. 95 AAAA ATOM 1290 0 ARG 136 36. 854 21. 445 67. 486 1. 00 20. 97 AAAA ATOM 1291 N TYR 137 36. 717 23. 684 67. 612 1. 00 17. 84 AAAA ATOM 1292 H TYR 137 37. 205 24. 511 67. 800 1. 00 0. 00 AAAA ATOM 1293 CA TYR 137 35. 289 23. 702 67. 369 1. 00 17. 54 AAAA ATOM 1294 CB TYR 137 34. 772 25. 129 67. 536 1. 00 18. 25 AAAA ATOM 1295 CG TYR 137 33. 262 25. 292 67. 445 1. 00 21. 44 AAAA ATOM 1296 CD1 TYR 137 32. 388 24. 288 67. 867 1. 00 22. 83 AAAA

ATOM 1297 CE1 TYR 137 30. 989 24. 460 67. 767 1. 00 24. 57 AAAA ATOM 1298 CD2 TYR 137 32. 712 26. 467 66. 931 1. 00 24. 44 AAAA ATOM 1299 CE2 TYR 137 31. 336 26. 645 66. 837 1. 00 23. 60 AAAA ATOM 1300 CZ TYR 137 30. 481 25. 645 67. 253 1. 00 23. 28 AAAA ATOM 1301 OH TYR 137 29. 124 25. 869 67. 158 1. 00 26. 64 AAAA ATOM 1302 HH TYR 137 28. 634 25. 119 67. 487 1. 00 0. 00 AAAA ATOM 1303 C TYR 137 34. 982 23. 136 65. 969 1. 00 19. 09 AAAA ATOM 1304 0 TYR 137 34. 284 22. 126 65. 862 1. 00 19. 43 AAAA ATOM 1305 N LEU 138 35. 682 23. 621 64. 939 1. 00 18. 53 AAAA ATOM 1306 H LEU 138 36. 295 24. 357 65. 103 1. 00 0. 00 AAAA ATOM 1307 CA LEU 138 35. 518 23. 068 63. 582 1. 00 20. 95 AAAA ATOM 1308 CB LEU 138 36. 472 23. 730 62. 586 1. 00 17. 16 AAAA ATOM 1309 CG LEU 138 36. 270 23. 242 61. 144 1. 00 17. 24 AAAA ATOM 1310 CD1 LEU 138 34. 88723. 658 60. 627 1. 00 18. 51 AAAA ATOM 1311 CD2 LEU 138 37. 357 23. 808 60. 262 1. 00 18. 65 AAAA ATOM 1312 C LEU 138 35. 679 21. 547 63. 480 1. 00 23. 49 AAAA ATOM 1313 0 LEU 138 34. 883 20. 888 62. 811 1. 00 23. 89 AAAA ATOM 1314 N GLU 139 36. 717 21. 001 64. 115 1. 00 23. 27 AAAA ATOM 1315 H GLU 139 37. 378 21. 603 64. 525 1. 00 0. 00 AAAA ATOM 1316 CA GLU 139 36. 884 19. 555 64. 225 1. 00 26. 59 AAAA ATOM 1317 CB GLU 139 38. 111 19. 221 65. 081 1. 00 31. 75 AAAA ATOM 1318 CG GLU 139 39. 429 19. 788 64. 559 1. 00 42. 46 AAAA ATOM 1319 CD GLU 139 40. 623 19. 513 65. 491 1. 00 47. 40 AAAA ATOM 1320 OE1 GLU 139 41. 176 18. 390 65. 443 1. 00 50. 41 AAAA ATOM 1321 OE2 GLU 139 41. 027 20. 429 66. 245 1. 00 50. 25 AAAA ATOM 1322 C GLU 139 35. 639 18. 897 64. 842 1. 00 27. 54 AAAA ATOM 1323 0 GLU 139 35. 147 17. 903 64. 318 1. 00 30. 37 AAAA ATOM 1324 N ASN 140 35. 090 19. 484 65. 904 1. 00 26. 57 AAAA ATOM 1325 H ASN 140 35. 529 20. 277 66. 280 1. 00 0. 00 AAAA ATOM 1326 CA ASN 140 33. 88418. 922 66. 545 1. 00 30. 15 AAAA ATOM 1327 CB ASN 140 33. 647 19. 558 67. 922 1. 00 32. 80 AAAA ATOM 1328 CG ASN 140 34. 831 19. 416 68. 854 1. 00 35. 71 AAAA ATOM 1329 OD1 ASN 140 35. 740 18. 621 68. 634 1. 00 37. 11 AAAA ATOM 1330 ND2 ASN 140 34. 828 20. 210 69. 906 1. 00 38. 04 AAAA ATOM 1331 HD21 ASN 140 34. 054 20. 795 70. 059 1. 00 0. 00 AAAA ATOM 1332 HD22ASN 140 35. 627 20. 136 70. 454 1. 00 0. 00 AAAA ATOM 1333 C ASN 140 32. 601 19. 106 65. 720 1. 00 30. 97 AAAA ATOM 1334 0 ASN 140 31. 654 18. 317 65. 819 1. 00 33. 89 AAAA ATOM 1335 N TYR 141 32. 524 20. 242 65. 041 1. 00 29. 95 AAAA ATOM 1336 H TYR 141 33. 363 20. 717 64. 917 1. 00 0. 00 AAAA ATOM 1337 CA TYR 141 31. 299 20. 738 64. 430 1. 00 28. 02 AAAA

ATOM 1338 CB TYR 141 31. 466 22. 222 64. 099 1. 00 25. 30 AAAA ATOM 1339 CG TYR 141 30. 191 22. 921 63. 727 1. 00 23. 02 AAAA ATOM 1340 CD1 TYR 141 29. 318 23. 352 64. 711 1. 00 24. 68 AAAA ATOM 1341 CE1 TYR 141 28. 155 24. 034 64. 396 1. 00 23. 87 AAAA ATOM 1342 CD2 TYR 141 29. 878 23. 186 62. 400 1. 00 17. 67 AAAA ATOM 1343 CE2 TYR 141 28. 722 23. 874 62. 073 1. 00 26. 80 AAAA ATOM 1344 CZ TYR 141 27. 863 24. 298 63. 083 1. 00 25. 22 AAAA ATOM 1345 OH TYR 141 26. 738 25. 024 62. 790 1. 00 27. 16 AAAA ATOM 1346 HH TYR 141 26. 724 25. 168 61. 847 1. 00 0. 00 AAAA ATOM 1347 C TYR 141 30. 968 19. 958 63. 162 1. 00 29. 46 AAAA ATOM 1348 0 TYR 141 31. 220 20. 422 62. 047 1. 00 28. 34 AAAA ATOM 1349 N ASP 142 30. 217 18. 882 63. 349 1. 00 30. 05 AAAA ATOM 1350 H ASP 142 29. 91318. 788 64. 276 1. 00 0. 00 AAAA ATOM 1351 CA ASP 142 29. 980 17. 888 62. 311 1. 00 33. 85 AAAA ATOM 1352 CB ASP 142 29. 123 16. 750 62. 880 1. 00 42. 00 AAAA ATOM 1353 CG ASP 142 29. 400 16. 473 64. 361 1. 00 49. 95 AAAA ATOM 1354 OD1 ASP 142 28. 696 17. 052 65. 230 1. 00 48. 78 AAAA ATOM 1355 OD2 ASP 142 30. 28815. 633 64. 648 1. 00 53. 91 AAAA ATOM 1356 C ASP 142 29. 308 18. 452 61. 047 1. 00 32. 97 AAAA ATOM 1357 0 ASP 142 29. 589 18. 017 59. 926 1. 00 31. 12 AAAA ATOM 1358 N ALA 143 28. 443 19. 442 61. 243 1. 00 30. 82 AAAA ATOM 1359 H ALA 143 28. 396 19. 832 62. 132 1. 00 0. 00 AAAA ATOM 1360 CA ALA 143 27. 622 19. 997 60. 176 1. 00 31. 33 AAAA ATOM 1361 CB ALA 143 26. 685 21. 056 60. 747 1. 00 30. 92 AAAA ATOM 1362 C ALA 143 28. 407 20. 577 58. 989 1. 00 32. 79 AAAA ATOM 1363 0 ALA 143 27. 900 20. 600 57. 869 1. 00 37. 58 AAAA ATOM 1364 N ILE 144 29. 628 21. 061 59. 215 1. 00 29. 28 AAAA ATOM 1365 H ILE 144 30. 015 21. 012 60. 119 1. 00 0. 00 AAAA ATOM 1366 CA ILE 144 30. 416 21. 591 58. 107 1. 00 27. 01 AAAA ATOM 1367 CB ILE 144 31. 344 22. 736 58. 568 1. 00 27. 54 AAAA ATOM 1368 CG2 ILE 144 32. 231 23. 223 57. 416 1. 00 27. 82 AAAA ATOM 1369 CG1 ILE 144 30. 485 23. 919 59. 024 1. 00 25. 26 AAAA ATOM 1370 CD ILE 144 31. 254 25. 121 59. 464 1. 00 24. 22 AAAA ATOM 1371 C ILE 144 31. 177 20. 463 57. 420 1. 00 29. 24 AAAA ATOM 1372 0 ILE 144 32. 313 20. 126 57. 774 1. 00 30. 95 AAAA ATOM 1373 N ARG 145 30. 45419. 795 56. 524 1. 00 28. 91 AAAA ATOM 1374 H ARG 145 29. 547 20. 114 56. 340 1. 00 0. 00 AAAA ATOM 1375 CA ARG 145 30. 895 18. 579 55. 834 1. 00 29. 33 AAAA ATOM 1376 CB ARG 145 30. 311 17. 342 56. 539 1. 00 30. 28 AAAA ATOM 1377 CG ARG 145 29. 988 16. 150 55. 646 0. 00 29. 38 AAAA ATOM 1378 CD ARG 145 31. 078 15. 091 55. 690 0. 00 29. 04 AAAA

ATOM 1379 NE ARG 145 31. 399 14. 683 57. 056 0. 00 28. 60 AAAA ATOM 1380 HE ARG 145 31. 308 15. 336 57. 782 0. 00 0. 00 AAAA ATOM 1381 CZ ARG 145 31. 833 13. 473 57. 396 0. 00 28. 42 AAAA ATOM 1382 NH1 ARG 145 32. 122 13. 208 58. 663 0. 00 28. 25 AAAA ATOM 1383 HH11ARG 145 32. 021 13. 923 59. 354 1. 00 0. 00 AAAA ATOM 1384 H12ARG 145 32.454 12.300 58.923 1.00 0. 00 AAAA ATOM 1385 NH2 ARG 145 31.974 12.524 56.479 0.00 28. 30 AAAA ATOM 1386 HH21 ARG 145 32. 31411. 623 56. 749 1. 00 0. 00 AAAA ATOM 1387 HH22 ARG 145 31.721 12.696 55.526 1.00 0. 00 AAAA ATOM 1388 C ARG 145 30. 355 18. 676 54. 415 1. 00 29. 88 AAAA ATOM 1389 0 ARG 145 29. 256 19. 167 54. 236 1. 00 31. 53 AAAA ATOM 1390 N VAL 146 31. 18718. 416 53. 415 1. 00 31. 91 AAAA ATOM 1391 H VAL 146 32.098 18.132 53.593 1.00 0. 00 AAAA ATOM 1392 CA VAL 146 30. 698 18. 434 52. 042 1. 00 37. 37 AAAA ATOM 1393 CB VAL 146 31. 845 18. 531 50. 978 1. 00 36. 83 AAAA ATOM 1394 CG1 VAL 146 32.686 19.774 51.208 1.00 33. 88 AAAA ATOM 1395 CG2 VAL 146 32. 71717. 285 50. 996 1. 00 36. 91 AAAA ATOM 1396 C VAL 146 29. 899 17. 155 51. 815 1. 00 44. 69 AAAA ATOM 1397 OT1 UAL 146 30. 301 16. 119 52. 395 1. 00 50. 73 AAAA ATOM 1398 OT2 VAL 146 28.808 17.228 51.203 1.00 51. 33 AAAA ATOM 1399 CB ASP 167 51. 716 18. 609 47. 898 1. 00 49. 54 BBBB ATOM 1400 CG ASP 167 53. 148 18. 936 47. 435 1. 00 55. 49 BBBB ATOM 1401 OD1 ASP 167 53. 852 17. 986 47. 033 1. 00 58. 95 BBBB ATOM 1402 OD2 ASP 167 53. 560 20. 123 47. 426 1. 00 57. 98 BBBB ATOM 1403 C ASP 167 50.546 20.690 48.673 1.00 38. 29 BBBB ATOM 1404 0 ASP 167 51. 079 21. 780 48. 786 1. 00 37. 35 BBBB ATOM 1405 HT1 ASP 167 49. 53118. 285 49. 508 1. 00 0. 00 BBBB ATOM 1406HT2ASP16750. 05519. 20350. 8361. 000. 00 BBBB ATOM 1407NASP16750. 38518. 62350. 0341. 0044. 61 BBBB ATOM 1408 HT3 ASP 167 50.902 17.811 50.403 1.00 0. 00 BBBB ATOM 1409 CA ASP 167 51. 272 19. 431 49. 130 1. 00 43. 35 BBBB ATOM 1410 N LEU 168 49. 330 20. 523 48. 152 1. 00 32. 28 BBBB ATOM 1411 H LEU 168 48. 99319. 646 47. 910 1. 00 0. 00 BBBB ATOM 1412 CA LEU 168 48. 483 21. 649 47. 735 1. 00 30. 48 BBBB ATOM 1413 CB LEU 168 47. 434 21. 174 46. 711 1. 00 29. 61 BBBB ATOM 1414 CG LEU 168 47. 519 21. 629 45. 234 1. 00 34. 32 BBBB ATOM 1415 CD1 LEU 168 48. 957 21. 832 44. 759 1. 00 27. 65 BBBB ATOM 1416 CD2 LEU 168 46. 80020. 592 44. 368 1. 00 27. 35 BBBB ATOM 1417 C LEU 168 47. 786 22. 321 48. 937 1. 00 27. 15 BBBB ATOM 1418 0 LEU 168 47. 365 21. 644 49. 867 1. 00 28. 57 BBBB ATOM 1419 N HIS 169 47. 71223. 649 48. 927 1. 00 22. 41 BBBB

ATOM 1420 H HIS 169 47. 989 24. 120 48. 109 1. 00 0. 00 BBBB ATOM 1421 CA HIS 169 47. 223 24. 415 50. 068 1. 00 16. 79 BBBB ATOM 1422 CB HIS 169 48. 425 24. 912 50. 889 1. 00 15. 93 BBBB ATOM 1423 CG HIS 169 48. 067 25. 462 52. 235 1. 00 13. 63 BBBB ATOM 1424 CD2 HIS 169 48. 301 26. 674 52. 792 1. 00 13. 31 BBBB ATOM 1425 ND1 HIS 169 47. 275 24. 780 53. 135 1. 00 19. 04 BBBB ATOM 1426 HD1 HIS 169 46. 915 23. 885 53. 069 1. 00 0. 00 BBBB ATOM 1427 CE1 HIS 169 47. 024 25. 550 54. 177 1. 00 15. 56 BBBB ATOM 1428 NE2 HIS 169 47. 641 26. 704 53. 996 1. 00 14. 61 BBBB ATOM 1429 HE2 HIS 169 47. 457 27. 477 54. 543 1. 00 0. 00 BBBB ATOM 1430 C HIS 169 46. 378 25. 605 49. 589 1. 00 15. 63 BBBB ATOM 1431 0 HIS 169 46. 655 26. 195 48. 551 1. 00 15. 56 BBBB ATOM 1432 N PHE 170 45. 336 25. 936 50. 347 1. 00 11. 37 BBBB ATOM 1433 H PHE 170 45. 080 25. 342 51. 078 1. 00 0. 00 BBBB ATOM 1434 CA PHE 170 44. 529 27. 119 50. 091 1. 00 13. 04 BBBB ATOM 1435 CB PHE 170 43. 034 26. 820 50. 263 1. 00 15. 00 BBBB ATOM 1436 CG PHE 170 42. 363 26. 294 49. 024 1. 00 13. 40 BBBB ATOM 1437 CD1 PHE 170 42. 008 24. 954 48. 930 1. 00 16. 80 BBBB ATOM 1438 CD2 PHE 170 41. 975 27. 160 48. 016 1. 00 17. 90 BBBB ATOM 1439 CE1 PHE 170 41. 253 24. 481 47. 862 1. 00 15. 73 BBBB ATOM 1440 CE2 PHE 170 41. 216 26. 692 46. 931 1. 00 19. 11 BBBB ATOM 1441 CZ PHE 170 40. 856 25. 352 46. 866 1. 00 14. 95 BBBB ATOM 1442 C PHE 170 44. 870 28. 218 51. 068 1. 00 12. 35 BBBB ATOM 1443 0 PHE 170 45. 009 27. 977 52. 273 1. 00 13. 04 BBBB ATOM 1444 N ILE 171 44. 908 29. 438 50. 562 1. 00 13. 08 BBBB ATOM 1445 H ILE 171 44. 992 29. 531 49. 589 1. 00 0. 00 BBBB ATOM 1446 CA ILE 171 44. 790 30. 602 51. 423 1. 00 17. 13 BBBB ATOM 1447 CB ILE 171 46. 115 31. 418 51. 506 1. 00 17. 52 BBBB ATOM 1448 CG2 ILE 171 47. 216 30. 540 52. 100 1. 00 18. 45 BBBB ATOM 1449 CG1 ILE 171 46. 520 31. 954 50. 133 1. 00 19. 39 BBBB ATOM 1450 CD ILE 171 47. 65332. 960 50. 180 1. 00 21. 31 BBBB ATOM 1451 C ILE 171 43. 646 31. 499 50. 942 1. 00 15. 82 BBBB ATOM 1452 0 ILE 171 43. 136 31. 317 49. 826 1. 00 15. 55 BBBB ATOM 1453 N ALA 172 43. 153 32. 332 51. 855 1. 00 14. 30 BBBB ATOM 1454 H ALA 172 43. 513 32. 279 52. 767 1. 00 0. 00 BBBB ATOM 1455 CA ALA 172 42. 126 33. 309 51. 554 1. 00 11. 34 BBBB ATOM 1456 CB ALA 172 40. 961 33. 159 52. 534 1. 00 12. 21 BBBB ATOM 1457 C ALA 172 42. 715 34. 705 51. 626 1. 00 13. 20 BBBB ATOM 1458 0 ALA 172 43. 537 35. 009 52. 500 1. 00 13. 74 BBBB ATOM 1459 N LEU 173 42. 324 35. 544 50. 670 1. 00 14. 10 BBBB ATOM 1460 H LEU 173 41. 732 35. 187 49. 978 1. 00 0. 00 BBBB

ATOM 1461 CA LEU 173 42. 744 36. 942 50. 630 1. 00 13. 28 BBBB ATOM 1462 CB LEU 173 43. 535 37. 208 49. 345 1. 00 14. 89 BBBB ATOM 1463 CG LEU 173 44. 958 36. 611 49. 358 1. 00 17. 25 BBBB ATOM 1464 CD1 LEU 173 45. 526 36. 424 47. 940 1. 00 14. 54 BBBB ATOM 1465 CD2 LEU 173 45. 847 37. 539 50. 191 1. 00 15, 04 BBBB ATOM 1466 C LEU 173 41. 502 37.827 50.688 1.00 16. 11 BBBB ATOM 1467 0 LEU 173 40. 568 37. 637 49. 907 1. 00 16. 45 BBBB ATOM 1468 N VAL 174 41. 425 38. 646 51. 736 1. 00 16. 31 BBBB ATOM 1469 H VAL 174 42. 209 38. 693 52. 325 1. 00 0. 00 BBBB ATOM 1470 CA VAL 174 40. 237 39. 448 52. 028 1. 00 17. 92 BBBB ATOM 1471 CB VAL 174 39. 351 38. 822 53. 157 1. 00 17. 86 BBBB ATOM 1472 CG1 VAL 174 38. 869 37.422 52.764 1.00 15. 30 BBBB ATOM 1473 CG2 VAL 174 40.130 38.787 54.480 1.00 16. 97 BBBB ATOM 1474 C VAL 174 40. 588 40.865 52.459 1.00 18. 53 BBBB ATOM 1475 0 VAL 174 41. 690 41. 149 52. 931 1. 00 17. 41 BBBB ATOM 1476 N HIS 175 39. 623 41. 751 52. 294 1. 00 18. 01 BBBB ATOM 1477 H HIS 175 38. 768 41. 427 51. 928 1. 00 0. 00 BBBB ATOM 1478 CA HIS 175 39. 784 43.137 52.667 1.00 22. 89 BBBB ATOM 1479 CB HIS 175 39. 178 44. 038 51. 590 1. 00 24. 46 BBBB ATOM 1480 CG HIS 175 38. 940 45. 443 52. 047 1. 00 28. 42 BBBB ATOM 1481 C02 HIS 175 39. 797 46.404 52.459 1.00 29. 90 BBBB ATOM 1482 ND1 HIS 175 37. 680 45. 987 52. 146 1. 00 29. 90 BBBB ATOM 1483 HD1 HIS 175 36. 868 45. 469 51. 941 1. 00 0. 00 BBBB ATOM 1484 CE1 HIS 175 37. 769 47.222 52.596 1.00 30. 93 BBBB ATOM 1485 NE2 HIS 175 39. 044 47. 499 52. 797 1. 00 31. 81 BBBB ATOM 1486 HE2 HIS 175 39. 397 48. 399 52. 952 1. 00 0. 00 BBBB ATOM 1487 C HIS 175 39. 090 43. 373 53. 994 1. 00 23. 68 BBBB ATOM 1488 0 HIS 175 37. 890 43.165 54.103 1.00 23. 93 BBBB ATOM 1489 N VAL 176 39. 852 43. 761 55. 011 1. 00 24. 25 BBBB ATOM 1490 H VAL 176 40. 832 43. 727 54. 897 1. 00 0. 00 BBBB ATOM 1491 CA VAL 176 39. 259 44. 145 56. 291 1. 00 25. 87 BBBB ATOM 1492 CB VAL 176 39. 486 43. 065 57. 389 1. 00 26. 10 BBBB ATOM 1493 CG1 VAL 176 38. 747 43. 442 58. 658 1. 00 23. 79 BBBB ATOM 1494 CG2 VAL 176 39. 012 41.709 56.905 1.00 23. 00 BBBB ATOM 1495 C VAL 176 39. 844 45.471 56.765 1.00 26. 17 BBBB ATOM 1496 0 VAL 176 41. 061 45. 661 56. 745 1. 00 25. 83 BBBB ATOM 1497 N ASP 177 38. 96146. 42357. 0641. 0030. 10 BBBB ATOM 1498 H ASP 177 38. 013 46. 197 56. 991 1. 00 0. 00 BBBB ATOM 1499 CA ASP 177 39. 348 47. 735 57. 593 1. 00 31. 23 BBBB ATOM 1500 CB ASP 177 39. 656 47. 634 59. 088 1. 00 37. 10 BBBB ATOM 1501 CG ASP 177 38.434 47.21 59.924 1.00 43. 92 BBBB

ATOM 1502 OD1 ASP 177 38. 610 46. 901 61. 115 1. 00 45. 60 BBBB ATOM 1503 OD2 ASP 177 37. 299 47. 249 59. 388 1. 00 48. 64 BBBB ATOM 1504 C ASP 177 40. 540 48. 339 56. 864 1. 00 30. 72 BBBB ATOM 1505 0 ASP 177 41. 580 48. 596 57. 472 1. 00 31. 89 BBBB ATOM 1506 N GLY 178 40. 456 48. 339 55. 535 1. 00 29. 77 BBBB ATOM 1507 H GLY 178 39. 689 47. 869 55. 161 1. 00 0. 00 BBBB ATOM 1508 CA GLY 178 41. 435 49. 035 54. 715 1. 00 30. 17 BBBB ATOM 1509 C GLY 178 42. 725 48. 298 54. 407 1. 00 28. 13 BBBB ATOM 1510 0 GLY 178 43. 607 48. 833 53. 738 1. 00 28. 30 BBBB ATOM 1511 N HIS 179 42. 842 47. 061 54. 877 1. 00 27. 85 BBBB ATOM 1512 H HIS 179 42. 13146. 661 55. 410 1. 00 0. 00 BBBB ATOM 1513 CA HIS 179 44. 031 46. 272 54. 588 1. 00 27. 46 BBBB ATOM 1514 CB HIS 179 44. 917 46. 186 55. 822 1. 00 31. 40 BBBB ATOM 1515 CG HIS 179 45. 299 47. 526 56. 364 1. 00 39. 75 BBBB ATOM 1516 CD2 HIS 179 46. 221 48. 424 55. 942 1. 00 39. 28 BBBB ATOM 1517 ND1 HIS 179 44. 541 48. 174 57. 317 1. 00 39. 91 BBBB ATOM 1518 HD1 HIS 179 43. 819 47. 792 57. 859 1. 00 0. 00 BBBB ATOM 1519 CE1 HIS 179 44. 971 49. 417 57. 448 1. 00 42. 46 BBBB ATOM 1520 NE2 HIS 179 45. 990 49. 592 56. 624 1. 00 43. 86 BBBB ATOM 1521 HE2 HIS 179 46. 444 50. 449 56. 500 1. 00 0. 00 BBBB ATOM 1522 C HIS 179 43. 726 44. 885 54. 074 1. 00 26. 07 BBBB ATOM 1523 0 HIS 179 42. 624 44. 362 54. 290 1. 00 24. 18 BBBB ATOM 1524 N LEU 180 44. 642 44. 397 53. 238 1. 00 23. 29 BBBB ATOM 1525 H LEU 180 45. 398 44. 952 52. 987 1. 00 0. 00 BBBB ATOM 1526 CA LEU 180 44. 582 43. 062 52. 646 1. 00 23. 82 BBBB ATOM 1527 CB LEU 180 45. 308 43. 066 51. 299 1. 00 22. 59 BBBB ATOM 1528 CG LEU 180 45. 434 41. 775 50. 491 1. 00 22. 52 BBBB ATOM 1529 CD1 LEU 180 44. 070 41. 212 50. 147 1. 00 22. 19 BBBB ATOM 1530 CD2 LEU 180 46. 206 42. 094 49. 223 1. 00 24. 25 BBBB ATOM 1531 C LEU 180 45. 208 42. 006 53. 560 1. 00 22. 99 BBBB ATOM 1532 0 LEU 180 46. 402 42. 059 53. 876 1. 00 23. 65 BBBB ATOM 1533 N TYR 181 44. 395 41. 055 53. 994 1. 00 20. 53 BBBB ATOM 1534 H TYR 181 43. 466 41. 057 53. 679 1. 00 0. 00 BBBB ATOM 1535 CA TYR 181 44. 867 40. 015 54. 896 1. 00 18. 31 BBBB ATOM 1536 CB TYR 181 43. 975 39. 921 56. 143 1. 00 16. 99 BBBB ATOM 1537 CG TYR 181 44. 141 41. 097 57. 092 1. 00 19. 91 BBBB ATOM 1538 CD1 TYR 181 43. 303 42. 210 57. 001 1. 00 21. 59 BBBB ATOM 1539 CE1 TYR 181 43. 493 43. 329 57. 820 1. 00 22. 12 BBBB ATOM 1540 CD2 TYR 181 45. 172 41. 126 58. 034 1. 00 19. 21 BBBB ATOM 1541 CE2 TYR 181 45. 361 42. 236 58. 863 1. 00 20. 75 BBBB ATOM 1542 CZ TYR 181 44.. 515 43. 332 58. 751 1. 00 21. 81 BBBB

ATOM 1543 OH TYR 181 44. 643 44. 415 59. 594 1. 00 21. 78 BBBB ATOM 1544 HH TYR 181 44. 032 45. 113 59. 325 1. 00 0. 00 BBBB ATOM 1545 C TYR 181 44. 903 38. 688 54. 188 1. 00 17. 40 BBBB ATOM 1546 0 TYR 181 44. 002 38. 360 53. 412 1. 00 16. 63 BBBB ATOM 1547 N GLU 182 46. 057 38. 049 54. 272 1. 00 15. 21 BBBB ATOM 1548 H GLU 182 46. 79138. 531 54. 688 1. 00 0. 00 BBBB ATOM 1549 CA GLU 182 46. 195 36. 674 53. 845 1. 00 13. 51 BBBB ATOM 1550 CB GLU 182 47. 642 36. 373 53. 429 1. 00 11. 85 BBBB ATOM 1551 CG GLU 182 47. 980 34. 891 53. 391 1. 00 11. 30 BBBB ATOM 1552 CD GLU 182 49. 468 34. 613 53. 230 1. 00 14. 98 BBBB ATOM 1553 OE1 GLU 182 50. 230 35. 561 52. 970 1. 00 15. 00 BBBB ATOM 1554 OE2 GLU 182 49. 865 33. 441 53. 342 1. 00 14. 93 BBBB ATOM 1555 C GLU 182 45. 847 35. 874 55. 071 1. 00 14. 99 BBBB ATOM 1556 0 GLU 182 46. 449 36. 065 56. 140 1. 00 15. 11 BBBB ATOM 1557 N LEU 183 44. 835 35. 028 54. 934 1. 00 12. 67 BBBB ATOM 1558 H LEU 183 44. 349 35. 063 54. 090 1. 00 0. 00 BBBB ATOM 1559 CA LEU 183 44. 445 34. 127 56. 002 1. 00 12. 90 BBBB ATOM 1560 CB LEU 183 42. 942 34. 265 56. 275 1. 00 15. 03 BBBB ATOM 1561 CG LEU 183 42. 456 35. 701 56. 541 1. 00 15. 56 BBBB ATOM 1562 CD1 LEU 183 40. 936 35. 730 56. 613 1. 00 15. 32 BBBB ATOM 1563 CD2 LEU 183 43. 039 36. 234 57. 858 1. 00 16. 89 BBBB ATOM 1564 C LEU 183 44. 82932. 687 55. 682 1. 00 11. 08 BBBB ATOM 1565 0 LEU 183 44. 329 32. 073 54. 757 1. 00 10. 71 BBBB ATOM 1566 N ASP 184 45. 836 32. 203 56. 387 1. 00 13. 16 BBBB ATOM 1567 H ASP 184 46. 219 32. 837 57. 037 1. 00 0. 00 BBBB ATOM 1568 CA ASP 184 46. 376 30. 859 56. 192 1. 00 14. 34 BBBB ATOM 1569 CB ASP 184 47. 759 30. 964 55. 527 1. 00 14. 33 BBBB ATOM 1570 CG ASP 184 48. 43229. 613 55. 334 1. 00 15. 44 BBBB ATOM 1571 OD1 ASP 184 49. 409 29. 556 54. 558 1. 00 16. 13 BBBB ATOM 1572 OD2 ASP 184 47. 999 28. 611 55. 936 1. 00 \14. 97 BBBB ATOM 1573 C ASP 184 46. 511 30. 220 57. 570 1. 00 16. 33 BBBB ATOM 1574 0 ASP 184 47. 346 30. 644 58. 356 1. 00 16. 05 BBBB ATOM 1575 N GLY 185 45. 728 29. 185 57. 850 1. 00 16. 42 BBBB ATOM 1576 H GLY 185 45. 154 28. 820 57. 141 1. 00 0. 00 BBBB ATOM 1577 CA GLY 185 45. 700 28. 623 59. 199 1. 00 17. 67 BBBB ATOM 1578 C GLY 185 46. 987 27. 929 59. 618 1. 00 18. 28 BBBB ATOM 1579 0 GLY 185 47. 13027. 51260. 7621. 0018. 70 BBBB ATOM 1580 N ARG 186 47. 867 27. 685 58. 650 1. 00 19. 25 BBBB ATOM 1581 H ARG 186 47. 535 27. 821 57. 748 1. 00 0. 00 BBBB ATOM 1582 CA ARG 186 49. 198 27. 155 58. 937 1. 00 19. 66 BBBB ATOM 1583 CB ARG 186 49. 89426. 714 57. 644 1. 00 17. 80 BBBB

ATOM 1584 CG ARG 186 49. 340 25. 429 57. 048 1. 00 21. 14 BBBB ATOM 1585 CD ARG 186 50. 144 24. 967 55. 843 1. 00 24. 27 BBBB ATOM 1586 NE ARG 186 51. 324 24. 196 56. 228 1. 00 28. 78 BBBB ATOM 1587 HE ARG 186 51. 195 23. 379 56. 760 1. 00 0. 00 BBBB ATOM 1588 CZ ARG 186 52. 575 24. 524 55. 907 1. 00 29. 73 BBBB ATOM 1589 NH1 ARG 186 53. 569 23. 710 56. 236 1. 00 28. 20 BBBB ATOM 1590 HH11 ARG 186 54. 505 23. 980 56. 016 1. 00 0. 00 BBBB ATOM 1591 HH12ARG 186 53. 390 22. 890 56. 781 1. 00 0. 00 BBBB ATOM 1592 NH2 ARG 186 52. 836 25. 644 55. 239 1. 00 27. 79 BBBB ATOM 1593 HH21 ARG 186 52. 060 26. 225 55. 039 1. 00 0. 00 BBBB ATOM 1594 HH22ARG 186 53. 769 25. 882 54. 957 1. 00 0. 00 BBBB ATOM 1595 C ARG 186 50. 068 28. 189 59. 644 1. 00 18. 52 BBBB ATOM 1596 0 ARG 186 51. 114 27. 871 60. 187 1. 00 23. 14 BBBB ATOM 1597 N LYS 187 49. 713 29. 450 59. 504 1. 00 16. 47 BBBB ATOM 1598 H LYS 187 48. 842 29. 675 59. 124 1. 00 0. 00. BBBB ATOM 1599 CA LYS 187 50. 543 30. 506 60. 036 1. 00 17. 08 BBBB ATOM 1600 CB LYS 187 50. 565 31. 671 59. 049 1. 00 13. 37 BBBB ATOM 1601 CG LYS 187 51. 244 31. 276 57. 737 1. 00 11. 69 BBBB ATOM 1602 CD LYS 187 51. 391 32. 462 56. 792 1. 00 12. 40 BBBB ATOM 1603 CE LYS 187 52. 035 32. 009 55. 491 1. 00 12. 88 BBBB ATOM 1604 NZ LYS 187 52. 331 33. 162 54. 604 1. 00 12. 97 BBBB ATOM 1605 HZ1 LYS 187 52. 773 33. 902 55. 169 1. 00 0. 00 BBBB ATOM 1606 HZ2 LYS 187 51. 46133. 563 54. 210 1. 00 0. 00 BBBB ATOM 1607 HZ3 LYS 187 52. 95132. 823 53. 846 1. 00 0. 00 BBBB ATOM 1608 C LYS 187 50. 068 30. 931 61. 426 1. 00 20. 11 BBBB ATOM 1609 0 LYS 187 48. 968 30.575 61.849 1.00 18. 37 BBBB ATOM 1610 N PRO 188 50. 942 31. 591 62. 199 1. 00 19. 32 BBBB ATOM 1611 CD PRO 188 52. 404 31. 644 62. 017 1. 00 19. 27 BBBB ATOM 1612 CA PRO 188 50. 522 31.951 63.565 1.00 20. 87 BBBB ATOM 1613 CB PRO 188 51. 831 32. 398 64. 248 1. 00 20. 84 BBBB ATOM 1614 CG PRO 188 52. 852 32.527 63.125 1.00 20. 92 BBBB ATOM 1615 C PRO 188 49. 442 33. 047 63. 618 1. 00 18. 51 BBBB ATOM 1616 0 PRO 188 48. 838 33. 303 64. 661 1. 00 18. 14 BBBB ATOM 1617 N PHE 189 49. 258 33. 744 62. 506 1. 00 16. 00 BBBB ATOM 1618 H PHE 189 49. 673 33. 425 61. 681 1. 00 0. 00 BBBB ATOM 1619 CA PHE 189 48. 441 34. 954 62. 474 1. 00 14. 73 BBBB ATOM 1620 CB PHE 189 49. 16136. 151 63. 126 1. 00 19. 72 BBBB ATOM 1621 CG PHE 189 50. 668 36.158 62.952 1.00 18. 33 BBBB ATOM 1622 CD1 PHE 189 51. 248 36. 237 61. 693 1. 00 19. 90 BBBB ATOM 1623 CD2 PHE 189 51. 502 36.056 64.061 1.00 24. 46 BBBB ATOM 1624 CE1 PHE 189 52. 632 36. 200 61. 537 1. 00 18. 74 BBBB

ATOM 1625 CE2 PHE 189 52. 891 36. 026 63. 913 1. 00 23. 39 BBBB ATOM 1626 CZ PHE 189 53. 449 36. 090 62. 650 1. 00 20. 37 BBBB ATOM 1627 C PHE 189 48. 102 35. 292 61. 035 1. 00 14. 51 BBBB ATOM 1628 0 PHE 189 48. 654 34. 694 60. 113 1. 00 14. 41 BBBB ATOM 1629 N PRO 190 47. 090 36. 150 60. 829 1. 00 18. 27 BBBB ATOM 1630 CD PRO 190 46. 101 36. 625 61. 817 1. 00 16. 54 BBBB ATOM 1631 CA PRO 190 46. 885 36. 773 59. 516 1. 00 15. 88 BBBB ATOM 1632 CB PRO 190 45. 744 37. 757 59. 764 1. 00 14. 74 BBBB ATOM 1633 CG PRO 190 45. 020 37. 200 60. 949 1. 00 13. 70 BBBB ATOM 1634 C PRO 190 48. 155 37. 495 59. 086 1. 00 18. 93 BBBB ATOM 1635 0 PRO 190 48. 944 37. 917 59. 937 1. 00 18. 46 BBBB ATOM 1636 N ILE 191 48. 385 37. 566 57. 781 1. 00 16. 14 BBBB.

ATOM 1637 H ILE 191 47. 81137. 011 57. 212 1. 00 0. 00 BBBB ATOM 1638 CA ILE 191 49. 478 38. 380 57. 235 1. 00 18. 56 BBBB ATOM 1639 CB ILE 191 50. 307 37. 590 56. 177 1. 00 15. 52 BBBB ATOM 1640 CG2 ILE 191 51. 435 38. 471 55. 632 1. 00 15. 36 BBBB ATOM 1641 CG1 ILE 191 50. 814 36. 270 56. 771 1. 00 14. 75 BBBB ATOM 1642 CD ILE 191 51. 668 36. 421 58. 031 1. 00 14. 66 BBBB ATOM 1643 C ILE 191 48. 922 39. 646 56. 584 1. 00 19. 70 BBBB ATOM 1644 0 ILE 191 48. 06239. 578 55. 691 1. 00 17. 72 BBBB ATOM 1645 N ASN 192 49. 352 40. 799 57. 091 1. 00 19. 71 BBBB ATOM 1646 H ASN 192 49. 955 40. 764 57. 864 1. 00 0. 00 BBBB ATOM 1647 CA ASN 192 48. 901 42. 083 56. 564 1. 00 22. 28 BBBB ATOM 1648 CB ASN 192 49. 056 43. 189 57. 615 1. 00 22. 85 BBBB ATOM 1649 CG ASN 192 48. 416 44. 496 57. 183 1. 00 20. 68 BBBB ATOM 1650 OD1 ASN 192 48. 436 44. 855 56. 012 1. 00 26. 53 BBBB ATOM 1651 ND2 ASN 192 47. 81445. 191 58. 122 1. 00 19. 89 BBBB ATOM 1652 HD21 ASN 192 47. 456 46. 053 57. 850 1. 00 0. 00 BBBB ATOM 1653 HD22ASN 192 47. 775 44. 794 59. 012 1. 00 0. 00 BBBB ATOM 1654 C ASN 192 49.725 42.422 55.327 1.00 23. 91 BBBB ATOM 1655 0 ASN 192 50. 945 42. 513 55. 405 1. 00 24. 94 BBBB ATOM 1656 N HIS 193 49. 070 42. 470 54. 169 1. 00 20. 91 BBBB ATOM 1657 H HIS 193 48. 105 42. 354 54. 248 1. 00 0. 00 BBBB ATOM 1658 CA HIS 193 49.767 42.707 52.912 1.00 20. 25 BBBB ATOM 1659 CB HIS 193 49. 263 41. 761 51. 834 1. 00 20. 01 BBBB ATOM 1660 CG HIS 193 49. 789 40. 375 51. 968 1. 00 17. 79 BBBB ATOM 1661 CD2 HIS 193 49. 220 39. 255 52. 470 1. 00 16. 56 BBBB ATOM 1662 ND1 HIS 193 51. 053 40. 007 51. 553 1. 00 18. 33 BBBB ATOM 1663 HD1 HIS 193 51. 743 40. 596 51. 162 1. 00 0. 00 BBBB ATOM 1664 CE1 HIS 193 51. 234 38. 723 51. 776 1. 00 20. 03 BBBB ATOM 1665 NE2 HIS 193 50. 135 38. 237 52. 335 1. 00 19. 35 BBBB

ATOM 1666 HE2 HIS 193 49. 908 37. 313 52. 494 1. 00 0. 00 BBBB ATOM 1667 C HIS 193 49. 639 44.132 52.422 1.00 22. 80 BBBB ATOM 1668 0 HIS 193 49. 923 44. 426 51. 253 1. 00 29. 84 BBBB ATOM 1669 N GLY 194 49. 166 45. 005 53. 296 1. 00 20. 96 BBBB ATOM 1670 H GLY 194 48. 902 44. 693 54. 183 1. 00 0. 00 BBBB ATOM 1671 CA GLY 194 49. 081 46. 400 52. 948 1. 00 24. 33 BBBB ATOM 1672 C GLY 194 47. 666 46. 870 52. 713 1. 00 26. 93 BBBB ATOM 1673 0 GLY 194 46. 698 46. 192 53. 033 1. 00 26. 69 BBBB ATOM 1674 N GLU 195 47. 552 48.092 52.226 1.00 29. 16 BBBB ATOM 1675 H GLU 195 48. 399 48. 549 52. 034 1. 00 0. 00 BBBB ATOM 1676 CA GLU 195 46. 259 48.704 52.028 1.00 33. 78 BBBB ATOM 1677 CB GLU 195 46. 428 50. 216 51. 875 1. 00 39. 83 BBBB ATOM 1678 CG GLU 195 47. 130 50. 868 53. 070 1. 00 50. 66 BBBB ATOM 1679 CD GLU 195 46. 813 52. 348 53. 202 1. 00 57. 04 BBBB ATOM 1680 OE1 GLU 195 46. 101 52. 716 54. 167 1. 00 59. 34 BBBB ATOM 1681 OE2 GLU 195 47. 273 53. 138 52. 344 1. 00 59. 02 BBBB ATOM 1682 C GLU 195 45.509 48.121 50.830 1.00 33. 75 BBBB ATOM 1683 0 GLU 195 46. 112 47. 615 49. 876 1. 00 32. 74 BBBB ATOM 1684 N THR 196 44. 185 48. 102 50. 947 1. 00 34. 10 BBBB ATOM 1685 H THR 196 43. 806 48. 428 51. 792 1. 00 0. 00 BBBB ATOM 1686 CA THR 196 43. 301 47. 694 49. 860 1. 00 31. 68 BBBB ATOM 1687 CB THR 196 43. 282 46. 156 49. 687 1. 00 29. 60 BBBB ATOM 1688 OG1 THR 196 42. 575 45. 819 48. 494 1. 00 29. 82 BBBB ATOM 1689 HG1 THR 196 43. 221 45.594 47.824 1.00 0. 00 BBBB ATOM 1690 CG2 THR 196 42. 599 45.489 50.864 1.00 28. 59 BBBB ATOM 1691 C THR 196 41. 882 48. 182 50. 156 1. 00 35. 53 BBBB ATOM 1692 O THR 196 415.87 48.676 51.258 1.00 37. 07 BBBB ATOM 1693 N SER 197 40. 978 47. 955 49. 210 1. 00 34. 92 BBBB ATOM 1694 H SER 197 41. 230 47. 386 48. 452 1. 00 0. 00 BBBB ATOM 1695 CA SER 197 39. 614 48. 467 49. 318 1. 00 35. 94 BBBB ATOM 1696 CB SER 197 39. 469 49. 780 48. 532 1. 00 35. 57 BBBB ATOM 1697 OG SER 197 39. 576 49. 545 47. 140 1. 00 38. 66 BBBB ATOM 1698 HG SER 197 40. 504 49. 351 46. 898 1. 00 0. 00 BBBB ATOM 1699 C SER 197 38. 645 47. 453 48. 752 1. 00 34. 85 BBBB ATOM 1700 0 SER 197 39. 049 46.547 48.023 1.00 35. 33 BBBB ATOM 1701 N ASP 198 37. 357 47. 688 48. 978 1. 00 35. 50 BBBB ATOM 1702 H ASP 198 37. 148 48. 444 49. 563 1. 00 0. 00 BBBB ATOM 1703 CA ASP 198 36. 314 46. 892 48. 341 1. 00 34. 12 BBBB ATOM 1704 CB ASP 198 34. 941 47. 470 48. 669 1. 00 38. 91 BBBB ATOM 1705 CG ASP 198 34. 511 47. 206 50. 096 1. 00 42. 65 BBBB ATOM 1706 OD1 ASP 198 35. 030 46. 260 50. 727 1. 00 41. 80 BBBB

ATOM 1707 0D2 ASP 198 33. 596 47. 923 50. 565 1. 00 48. 05 BBBB ATOM 1708 C ASP 198 36.490 46.878 46.821 1.00 32. 19 BBBB ATOM 1709 0 ASP 198 36. 313 45. 851 46. 159 1. 00 30. 03 BBBB ATOM 1710 N GLU 199 36. 900 48. 018 46. 283 1. 00 31. 22 BBBB ATOM 1711 H GLU 199 37. 225 48. 737 46. 860 1. 00 0. 00 BBBB ATOM 1712 CA GLU 199 36. 906 48. 216 44. 845 1. 00 32. 54 BBBB ATOM 1713 CB GLU 199 36. 89649. 714 44. 535 1. 00 35. 99 BBBB <BR> <BR> <BR> ATOM 1714 CG GLU 199 36. 693 50. 048 43. 064 0. 00 35. 94 BBBB ATOM 1715 CD GLU 199 36. 794 51. 537 42. 786 0. 00 36. 71 BBBB ATOM 1716 OE1 GLU 199 35. 736 52. 191 42. 663 0. 00 36. 92 BBBB ATOM 1717 OE2 GLU 199 37. 927 52. 049 42. 678 0. 00 36. 88 BBBB ATOM 1718 C GLU 199 38. 102 47. 549 44. 182 1. 00 33. 23 BBBB ATOM 1719 0 GLU 199 38. 022 47. 094 43. 042 1. 00 35. 95 BBBB ATOM 1720 N THR 200 39. 211 47. 466 44. 900 1. 00 31. 19 BBBB ATOM 1721 H THR 200 39. 195 47. 740 45. 842 1. 00 0. 00 BBBB ATOM 1722 CA THR 200 40. 448 47. 014 44. 286 1. 00 29. 88 BBBB ATOM 1723 CB THR 200 41. 550 48. 032 44. 504 1. 00 30. 21 BBBB ATOM 1724 OG1 THR 200 41. 741 48. 226 45. 912 1. 00 33. 62 BBBB ATOM 1725 HG1 THR 200 42. 647 47. 966 46. 081 1. 00 0. 00 BBBB ATOM 1726 CG2 THR 200 41. 169 49. 349 43. 883 1. 00 32. 46 BBBB ATOM 1727 C THR 200 40. 929 45. 671 44. 820 1. 00 29. 23 BBBB ATOM 1728 0 THR 200 42. 026 45. 222 44. 478 1. 00 29. 48 BBBB ATOM 1729 N LEU 201 40.112 45.022 45.645 1.00 26. 09 BBBB ATOM 1730 H LEU 201 39. 262 45. 438 45. 894 1. 00 0. 00 BBBB ATOM 1731 CA LEU 201 40.495 40.741 46.234 1.00 24. 40 BBBB ATOM 1732 CB LEU 201 39. 347 43. 148 47. 081 1. 00 23. 41 BBBB ATOM 1733 CG LEU 201 39.696 41.793 47.723 1.00 22. 38 BBBB ATOM 1734 CD1 LEU 201 40.856 41.971 48.697 1.00 19. 43 BBBB ATOM 1735 CD2 LEU 201 38. 51841. 208 48. 436 1. 00 20. 99 BBBB ATOM 1736 C LEU 201 41.004 42.699 45.222 1.00 24. 10 BBBB ATOM 1737 O LEU 201 42.039 42.067 45.58 1.00 21. 46 BBBB ATOM 1738 N LEU 202 40. 311 42. 538 44. 092 1. 00 23. 29 BBBB ATOM 1739 H LEU 202 39. 506 43. 062 43. 967 1. 00 0. 00 BBBB ATOM 1740 CA LEU 202 40. 734 41. 578 43. 076 1. 00 23. 58 BBBB ATOM 1741 CB LEU 202 39. 769 41. 570 41. 881 1. 00 22. 67 BBBB ATOM 1742 CG LEU 202 40. 12340. 532 40. 796 1. 00 23. 83 BBBB ATOM 1743 CD1 LEU 202 40.060 39.135 41.378 1.00 23. 97 BBBB ATOM 1744 CD2 LEU 202 39. 18440. 629 39. 609 1. 00 24. 35 BBBB ATOM 1745 C LEU 202 42. 167 41. 851 42. 583 1. 00 24. 21 BBBB ATOM 1746 O LEU 202 43.008 40.966 42.636 1.00 25. 26 BBBB ATOM 1747 N GLU 203 42.466 43.102 42.246 1.00 24. 33 BBBB

ATOM 1748 H GLU 203 41. 743 43. 756 42. 344 1. 00 0. 00 BBBB ATOM 1749 CA GLU 203 43. 797 43.484 41.769 1.00 26. 76 BBBB ATOM 1750 CB GLU 203 43. 822 44. 942 41. 347 1. 00 30. 49 BBBB ATOM 1751 CG GLU 203 43. 153 45. 233 40. 052 1. 00 44. 95 BBBB ATOM 1752 CD GLU 203 41. 698 44. 838 40. 070 1. 00 53. 45 BBBB ATOM 1753 OE1 GLU 203 40. 954 45. 332 40. 954 1. 00 53. 33 BBBB ATOM 1754 OE2 GLU 203 41. 329 43. 967 39. 245 1. 00 59. 22 BBBB ATOM 1755 C GLU 203 44. 853 43. 314 42. 844 1. 00 25. 56 BBBB ATOM 1756 0 GLU 203 45. 928 42.792 42.585 1.00 26. 26 BBBB ATOM 1757 N ASP 204 44. 567 43.846 44.027 1.00 24. 51 BBBB ATOM 1758 H ASP 204 43. 705 44.302 44.116 1.00 0. 00 BBBB ATOM 1759 CA ASP 204 45. 501 43. 796 45. 156 1. 00 26. 31 BBBB ATOM 1760 CB ASP 204 44.968 44.665 46.303 1.00 24. 27 BBBB ATOM 1761 CG ASP 204 44. 849 46. 143 45. 911 1. 00 30. 80 BBBB ATOM 1762 OD1 ASP 204 45. 337 46. 520 44. 820 1. 00 31. 50 BBBB ATOM 1763 OD2 ASP 204 44.263 46.935 46.682 1.00 30. 67 BBBB ATOM 1764 C ASP 204 45. 807 42.370 45.646 1.00 25. 41 BBBB ATOM 1765 0 ASP 204 46. 962 42. 039 45. 946 1. 00 25. 28 BBBB ATOM 1766 N ALA 205 44. 800 41. 501 45. 617 1. 00 21. 59 BBBB ATOM 1767 H ALA 205 43. 910 41. 827 45. 372 1. 00 0. 00 BBBB ATOM 1768 CA ALA 205 44. 993 40. 103 45. 971 1. 00 20. 94 BBBB ATOM 1769 CB ALA 205 43. 635 39. 417 46. 210 1. 00 19. 51 BBBB ATOM 1770 C ALA 205 45. 792 39. 335 44. 919 1. 00 23. 38 BBBB ATOM 1771 0 ALA 205 46. 581 38. 449 45. 245 1. 00 23. 30 BBBB ATOM 1772 N ILE 206 45. 561 39. 641 43. 648 1. 00 24. 50 BBBB ATOM 1773 H ILE 206 44. 86140. 303 43. 440 1. 00 0. 00 BBBB ATOM 1774 CA ILE 206 46. 316 38. 996 42. 577 1. 00 23. 48 BBBB ATOM 1775 CB ILE 206 45. 614 39. 219 41. 198 1. 00 25. 93 BBBB ATOM 1776 CG2 ILE 206 46.548 38.880 40.046 1.00 28. 83 BBBB ATOM 1777 CG1 ILE 206 44. 306 38. 403 41. 140 1. 00 24. 14 BBBB ATOM 1778 CD ILE 206 44. 460 36. 916 41. 168 1. 00 22. 80 BBBB ATOM 1779 C ILE 206 47. 794 39. 462 42. 556 1. 00 21. 88 BBBB ATOM 1780 0 ILE 206 48. 690 38. 647 42. 356 1. 00 22. 55 BBBB ATOM 1781 N GLU 207 48. 053 40.704 42.952 1.00 23. 56 BBBB ATOM 1782 H GLU 207 47. 305 41. 330 43. 060 1. 00 0. 00 BBBB ATOM 1783 CA GLU 207 49. 433 41. 154 43. 183 1. 00 24. 83 BBBB ATOM 1784 CB GLU 207 49. 46242. 580 43. 716 1. 00 28. 57 BBBB ATOM 1785 CG GLU 207 48. 898 43. 637 42. 782 1. 00 38. 93 BBBB ATOM 1786 CD GLU 207 49. 151 43. 327 41. 327 1. 00 46. 17 BBBB ATOM 1787 OE1 GLU 207 50. 338 43.204 40.949 1.00 46. 72 BBBB ATOM 1788 OE2 GLU 207 48. 157 43. 174 40. 573 1. 00 53. 27 BBBB

ATOM 1789 C GLU 207 50. 150 40. 256 44. 176 1. 00 26. 10 BBBB ATOM 1790 0 GLU 207 51. 250 39. 775 43. 910 1. 00 24. 31 BBBB ATOM 1791 N VAL 208 49. 502 39. 990 45. 311 1. 00 26. 00 BBBB ATOM 1792 H VAL 208 48. 670 40. 493 45. 482 1. 00 0. 00 BBBB ATOM 1793 CA VAL 208 50. 015 39. 009 46. 273 1. 00 24. 95 BBBB ATOM 1794 CB VAL 208 49. 129 38. 946 47. 549 1. 00 24. 26 BBBB ATOM 1795 CG1 VAL 208 49. 612 37. 844 48. 496 1. 00 23. 70 BBBB ATOM 1796 CG2 VAL 208 49. 155 40. 275 48. 244 1. 00 21. 67 BBBB ATOM 1797 C VAL 208 50. 151 37. 597 45. 674 1. 00 26. 78 BBBB ATOM 1798 0 VAL 208 51. 154 36. 911 45. 916 1. 00 29. 35 BBBB ATOM 1799 N CYS 209 49. 183 37. 171 44. 863 1. 00 22. 50 BBBB ATOM 1800 H CYS 209 48. 377 37. 730 44. 776 1. 00 0. 00 BBBB ATOM 1801 CA CYS 209 49. 296 35. 871 44. 203 1. 00 25. 82 BBBB ATOM 1802 CB CYS 209 48. 036 35. 546 43. 395 1. 00 26. 60 BBBB ATOM 1803 SG CYS 209 46. 543 35. 387 44. 399 1. 00 28. 29 BBBB ATOM 1804 C CYS 209 50. 518 35. 796 43. 277 1. 00 26. 91 BBBB ATOM 1805 0 CYS 209 51. 212 34. 772 43. 229 1. 00 25. 85 BBBB ATOM 1806 N LYS 210 50. 766 36. 875 42. 537 1. 00 29. 99 BBBB ATOM 1807 H LYS 210 50. 179 37. 646 42. 629 1. 00 0. 00 BBBB ATOM 1808 CA LYS 210 51. 919 36. 947 41. 631 1. 00 34. 45 BBBB ATOM 1809 CB LYS 210 51. 820 38. 188 40. 737 1. 00 33. 88 BBBB ATOM 1810 CG LYS 210 50. 789 38. 051 39. 625 1. 00 39. 79 BBBB ATOM 1811 CD LYS 210 50. 793 39. 254 38. 687 1. 00 40. 27 BBBB ATOM 1812 CE LYS 210 49. 886 40. 351 39. 183 1. 00 37. 63 BBBB ATOM 1813 NZ LYS 210 50. 066 41. 602 38. 404 1. 00 42. 21 BBBB ATOM 1814 HZ1 LYS 210 49. 737 41. 477 37. 425 1. 00 0. 00 BBBB ATOM 1815 HZ2 LYS 210 51. 073 41. 858 38. 408 1. 00 0. 00 BBBB ATOM 1816 HZ3 LYS 210 49. 517 42. 363 38. 853 1. 00 0. 00 BBBB ATOM 1817 C LYS 210 53. 255 36. 951 42. 395 1. 00 35. 34 BBBB ATOM 1818 0 LYS 210 54. 226 36. 338 41. 956 1. 00 37. 89 BBBB ATOM 1819 N LYS 211 53. 252 37. 513 43. 601 1. 00 33. 64 BBBB ATOM 1820 H LYS 211 52. 463 38. 041 43. 867 1. 00 0. 00 BBBB ATOM 1821 CA LYS 211 54. 402 37. 428 44. 485 1. 00 33. 59 BBBB ATOM 1822 CB LYS 211 54. 171 38. 308 45. 710 1. 00 37. 75 BBBB ATOM 1823 CG LYS 211 54. 711 39. 721 45. 563 1. 00 43. 42 BBBB ATOM 1824 CD LYS 211 54. 10940. 692 46. 592 1. 00 46. 72 BBBB ATOM 1825 CE LYS 211 53. 93340. 053 47. 968 1. 00 51. 43 BBBB ATOM 1826 NZ LYS 211 53. 323 41. 004 48. 941 1. 00 56. 76 BBBB ATOM 1827 HZ1 LYS 211 52. 474 41. 435 48. 539 1. 00 0. 00 BBBB ATOM 1828 HZ2 LYS 211 54. 006 41. 758 49. 162 1. 00 0. 00 BBBB ATOM 1829 HZ3 LYS 211 53. 063 40. 507 49. 815 1. 00 0. 00 BBBB

ATOM 1830 C LYS 211 54. 693 35. 984 44. 917 1. 00 34. 73 BBBB ATOM 1831 0 LYS 211 55. 852 35. 548 44. 921 1. 00 35. 11 BBBB ATOM 1832 N PHE 212 53. 643 35. 231 45. 243 1. 00 29. 19 BBBB ATOM 1833 H PHE 212 52. 776 35. 679 45. 325 1. 00 0. 00 BBBB ATOM 1834 CA PHE 212 53. 775 33. 795 45. 490 1. 00 26. 68 BBBB ATOM 1835 CB PHE 212 52. 394 33. 176 45. 751 1. 00 25. 71 BBBB ATOM 1836 CG PHE 212 51. 936 33. 288 47. 180 1. 00 21. 00 BBBB ATOM 1837 CD1 PHE 212 51. 76634. 545 47. 774 1. 00 19. 39 BBBB ATOM 1838 CD2 PHE 212 51. 759 32. 137 47. 950 1. 00 21. 10 BBBB ATOM 1839 CE1 PHE 212 51. 434 34. 655 49. 131 1. 00 20. 27 BBBB ATOM 1840 CE2 PHE 212 51.426 32.226 49.303 1.00 21. 64 BBBB ATOM 1841 CZ PHE 212 51. 266 33. 497 49. 896 1. 00 19. 69 BBBB ATOM 1842 C PHE 212 54. 438 33. 069 44. 308 1. 00 27. 94 BBBB ATOM 1843 0 PHE 212 55. 379 32. 289 44. 469 1. 00 29. 82 BBBB ATOM 1844 N MET 213 53. 942 33. 347 43. 114 1. 00 28. 49 BBBB ATOM 1845 H MET 213 53. 195 33. 982 43. 073 1. 00 0. 00 BBBB ATOM 1846 CA MET 213 54. 439 32. 709 41. 909 1. 00 31. 13 BBBB ATOM 1847 CB MET 213 53. 534 33. 080 40. 748 1. 00 31. 76 BBBB ATOM 1848 CG MET 213 52. 139 32. 540 40. 911 1. 00 32. 92 BBBB ATOM 1849 SD MET 213 51. 14432. 918 39. 495 1. 00 39. 38 BBBB ATOM 1850 CE MET 213 51. 471 31. 457 38. 429 1. 00 35. 56 BBBB ATOM 1851 C MET 213 55. 888 33. 089 41. 596 1. 00 32. 08 BBBB ATOM 1852 O MET 213 56.729 32.220 41.440 1.00 30. 06 BBBB ATOM 1853 N GLU 214 56. 192 34. 380 41. 688 1. 00 36. 22 BBBB ATOM 1854 H GLU 214 55. 459 34. 995 41. 879 1. 00 0. 00 BBBB ATOM 1855 CA GLU 214 57. 558 34. 879 41. 512 1. 00 42. 62 BBBB ATOM 1856 CB GLU 214 57. 613 36. 380 41. 817 1. 00 45. 96 BBBB ATOM 1857 CG GLU 214 57. 738 37. 265 40. 579 1. 00 54. 19 BBBB ATOM 1858 CD GLU 214 56. 753 38. 427 40. 570 1. 00 59. 49 BBBB ATOM 1859 DE1 GLU 214 56. 618 39. 120 41. 609 1. 00 62. 81 BBBB ATOM 1860 OE2 GLU 214 56. 119 38. 648 39. 513 1. 00 61. 93 BBBB ATOM 1861 C GLU 214 58. 572 34. 144 42. 392 1. 00 44. 13 BBBB ATOM 1862 0 GLU 214 59. 653 33. 783 41. 938 1. 00 46. 23 BBBB ATOM 1863 N ARG 215 58. 188 33. 867 43. 633 1. 00 44. 81 BBBB ATOM 1864 H ARG 215 57. 295 34. 167 43. 910 1. 00 0. 00 BBBB ATOM 1865 CA ARG 215 59. 052 33. 164 44. 573 1. 00 47. 12 BBBB ATOM 1866 CB ARG 215 58. 419 33. 174 45. 959 1. 00 49. 23 BBBB ATOM 1867 CG ARG 215 58. 442 34. 512 46. 634 1. 00 54. 62 BBBB ATOM 1868 CD ARG 215 57. 749 34. 429 47. 970 1. 00 59. 28 BBBB ATOM 1869 NE ARG 215 57. 032 35. 662 48. 269 1. 00 63. 64 BBBB ATOM 1870 HE ARG 215 57. 427 36. 515 47. 986 1. 00 0. 00 BBBB

ATOM 1871 CZ ARG 215 55. 866 35. 700 48. 902 1. 00 66. 38 BBBB ATOM 1872 NH1 ARG 215 55. 281 36. 866 49. 147 1. 00 69. 59 BBBB ATOM 1873 HH11ARG 215 55. 682 37. 708 48. 788 1. 00 0. 00 BBBB ATOM 1874 HH12ARG 215 54. 403 36. 895 49. 629 1. 00 0. 00 BBBB ATOM 1875 NH2 ARG 215 55. 298 34. 569 49. 314 1. 00 66. 82 BBBB ATOM 1876 HH21 ARG 215 54. 426 34. 601 49. 803 1. 00 0. 00 BBBB ATOM 1877 HH22ARG 215 55. 71133. 685 49. 101 1. 00 0. 00 BBBB ATOM 1878 C ARG 215 59. 321 31. 719 44. 166 1. 00 48. 10 BBBB ATOM 1879 0 ARG 215 60. 245 31. 077 44. 670 1. 00 49. 48 BBBB ATOM 1880 N ASP 216 58. 422 31. 164 43. 369 1. 00 47. 98 BBBB ATOM 1881 H ASP 216 57. 679 31. 721 43. 049 1. 00 0. 00 BBBB ATOM 1882 CA ASP 216 58. 530 29. 771 42. 972 1. 00 47. 13 BBBB ATOM 1883 CB ASP 216 57. 473 28. 944 43. 711 1. 00 47. 78 BBBB ATOM 1884 CG ASP 216 57. 963 27. 553 44. 093 1. 00 50. 19 BBBB ATOM 1885 OD1 ASP 216 58. 843 26. 996 43. 395 1. 00 51. 39 BBBB ATOM 1886 OD2 ASP 216 57. 431 26. 985 45. 072 1. 00 48. 92 BBBB ATOM 1887 C ASP 216 58. 355 29. 651 41. 458 1. 00 47. 63 BBBB ATOM 1888 0 ASP 216 57. 382 29. 067 40. 979 1. 00 48. 75 BBBB ATOM 1889 N PRO 217 59. 337 30. 147 40. 685 1. 00 47. 00 BBBB ATOM 1890 CD PRO 217 60. 664 30. 580 41. 150 1. 00 46. 27 BBBB ATOM 1891 CA PRO 217 59. 102 30. 558 39. 299 1. 00 46. 24 BBBB ATOM 1892 CB PRO 217 60. 344 31. 373 38. 953 1. 00 45. 55 BBBB ATOM 1893 CG PRO 217 60. 942 31. 740 40. 263 1. 00 45. 13 BBBB ATOM 1894 C PRO 217 58. 910 29. 393 38. 335 1. 00 47. 66 BBBB ATOM 1895 0 PRO 217 58. 407 29. 573 37. 230 1. 00 50. 76 BBBB ATOM 1896 N ASP 218 59. 325 28. 200 38. 742 1. 00 48. 45 BBBB ATOM 1897 H ASP 218 59. 857 28. 156 39. 563 1. 00 0. 00 BBBB ATOM 1898 CA ASP 218 59. 133 27. 016 37. 912 1. 00 53. 00 BBBB ATOM 1899 CB ASP 218 60. 313 26. 054 38. 073 1. 00 59. 27 BBBB ATOM 190D CG ASP 218 61. 401 26. 286 37. 038 1. 00 62. 24 BBBB ATOM 1901 OD1 ASP 218 62. 264 27. 163 37. 278 1. 00 61. 84 BBBB ATOM 1902 OD2 ASP 218 61. 384 25. 592 35. 990 1. 00 64. 66 BBBB ATOM 1903 C ASP 218 57. 847 26. 266 38. 214 1. 00 54. 01 BBBB ATOM 1904 0 ASP 218 57. 470 25. 352 37. 476 1. 00 55. 95 BBBB ATOM 1905 N GLU 219 57. 221 26. 606 39. 339 1. 00 54. 49 BBBB ATOM 1906 H GLU 219 57. 552 27. 392 39. 808 1. 00 0. 00 BBBB ATOM 1907 CA GLU 219 55. 990 25. 951 39. 789 1. 00 53. 25 BBBB ATOM 1908 CB GLU 219 55. 744 26. 201 41. 285 1. 00 52. 75 BBBB ATOM 1909 CG GLU 219 56. 022 25. 007 42. 175 1. 00 53. 61 BBBB ATOM 1910 CD GLU 219 55. 203 23. 766 41. 812 1. 00 58. 48 BBBB ATOM 1911 OE1 GLU 219 54. 269 23. 854 40. 975 1. 00 58. 85 BBBB

ATOM 1912 OE2 GLU 219 55. 486 22. 689 42. 389 1. 00 59. 45 BBBB ATOM 1913 C GLU 219 54. 766 26. 393 39. 001 1. 00 51. 04 BBBB ATOM 1914 0 GLU 219 54. 415 27. 573 38. 987 1. 00 49. 15 BBBB ATOM 1915 N LEU 220 54. 077 25. 419 38. 421 1. 00 50. 75 BBBB ATOM 1916 H LEU 220 54. 442 24. 515 38. 500 1. 00 0. 00 BBBB ATOM 1917 CA LEU 220 52. 915 25. 686 37. 578 1. 00 51. 68 BBBB ATOM 1918 CB LEU 220 52. 817 24. 622 36. 484 1. 00 54. 60 BBBB ATOM 1919 CG LEU 220 53. 302 24. 997 35. 084 1. 00 58. 03 BBBB ATOM 1920 CD1 LEU 220 52. 102 25. 048 34. 146 1. 00 59. 62 BBBB ATOM 1921 CD2 LEU 220 54. 038 26. 341 35. 107 1. 00 60. 10 BBBB ATOM 1922 C LEU 220 51. 600 25. 726 38. 357 1. 00 49. 77 BBBB ATOM 1923 0 LEU 220 50. 638 26. 363 37. 929 1. 00 48. 89 BBBB ATOM 1924 N ARG 221 51. 605 25. 116 39. 540 1. 00 47. 79 BBBB ATOM 1925 H ARG 221 52. 470 25. 007 39. 968 1. 00 0. 00 BBBB ATOM 1926 CA ARG 221 50. 377 24. 679 40. 217 1. 00 45. 16 BBBB ATOM 1927 CB ARG 221 50. 680 23. 414 41. 049 1. 00 48. 94 BBBB ATOM 1928 CG ARG 221 51. 461 22. 326 40. 282 1. 00 51. 97 BBBB ATOM 1929 CD ARG 221 52. 261 21. 403 41. 202 1. 00 59. 14 BBBB ATOM 1930 NE ARG 221 51. 422 20. 383 41. 840 1. 00 71. 38 BBBB ATOM 1931 HE ARG 221 50. 867 20. 658 42. 597 1. 00 20. 00 BBBB ATOM 1932 CZ ARG 221 51. 331 19. 109 41. 447 1. 00 75. 94 BBBB ATOM 1933 NH1 ARG 221 50. 48618. 27942. 0611. 0075. 00 BBBB ATOM 1934 HH11 ARG 221 49. 905 18. 611 42. 804 1. 00 0. 00 BBBB ATOM 1935 HH12ARG 221 50. 41717. 331 41. 752 1. 00 0. 00 BBBB ATOM 1936 NH2 ARG 221 52. 093 18. 651 40. 453 1. 00 77. 87 BBBB ATOM 1937 HH21 ARG 221 52. 745 19. 258 39. 998 1. 00 0. 00 BBBB ATOM 1938 HH22ARG 221 52. 024 17. 696 40. 164 1. 00 0. 00 BBBB ATOM 1939 C ARG 221 49. 715 25. 774 41. 085 1. 00 38. 76 BBBB ATOM 1940 0 ARG 221 49. 451 25. 580 42. 271 1. 00 33. 96 BBBB ATOM 1941 N PHE 222 49. 467 26. 925 40. 469 1. 00 32. 85 BBBB ATOM 1942 H PHE 222 49. 676 27. 003 39. 511 1. 00 0. 00 BBBB ATOM 1943 CA PHE 222 48. 830 28. 062 41. 123 1. 00 31. 95 BBBB ATOM 1944 CB PHE 222 49. 698 29. 321 40. 980 1. 00 35. 29 BBBB ATOM 1945 CG PHE 222 50. 951 29. 306 41. 830 1. 00 40. 24 BBBB ATOM 1946 CD1 PHE 222 52. 100 28. 634 41. 394 1. 00 39. 81 BBBB ATOM 1947 CD2 PHE 222 50. 958 29. 904 43. 095 1. 00 38. 34 BBBB ATOM 1948 CE1 PHE 222 53. 23428. 540 42. 212 1. 00 42. 53 BBBB ATOM 1949 CE2 PHE 222 52. 087 29. 818 43. 927 1. 00 40. 72 BBBB ATOM 1950 CZ PHE 222 53. 229 29. 131 43. 485 1. 00 41. 46 BBBB ATOM 1951 C PHE 222 47. 485 28. 312 40. 440 1. 00 31. 88 BBBB ATOM 1952 0 PHE 222 47. 438 28. 497 39. 233 1. 00 30. 41 BBBB

ATOM 1953 N ASN 223 46. 393 28. 244 41. 195 1. 00 25. 46 BBBB ATOM 1954 H ASN 223 46. 518 28. 097 42. 159 1. 00 0. 00 BBBB ATOM 1955 CA ASN 223 45. 057 28. 452 40. 632 1. 00 22. 95 BBBB ATOM 1956 CB ASN 223 44. 389 27. 109 40. 377 1. 00 22. 96 BBBB ATOM 1957 CG ASN 223 45. 19026. 234 39. 440 1. 00 30. 10 BBBB ATOM 1958 OD1 ASN 223 44. 896 26. 142 38. 249 1. 00 39. 00 BBBB ATOM 1959 ND2 ASN 223 46.227 25.602 39.965 1.00 34. 21 BBBB ATOM 1960 HD21 ASN 223 46. 685 24. 977 39. 365 1. 00 0. 00 BBBB ATOM 1961 HD22ASN 223 46. 478 25. 799 40. 881 1. 00 0. 00 BBBB ATOM 1962 C ASN 223 44. 211 29. 248 41. 608 1. 00 20. 90 BBBB ATOM 1963 0 ASN 223 44. 150 28. 908 42. 784 1. 00 24. 24 BBBB ATOM 1964 N ALA 224 43. 598 30. 324 41. 148 1. 00 19. 58 BBBB ATOM 1965 H ALA 224 43. 709 30. 554 40. 197 1. 00 0. 00 BBBB ATOM 1966 CA ALA 224 42. 785 31. 162 42. 030 1. 00 20. 81 BBBB ATOM 1967 CB ALA 224 43. 349 32. 588 42. 086 1. 00 17. 03 BBBB ATOM 1968 C ALA 224 41. 292 31. 199 41. 651 1. 00 22. 36 BBBB ATOM 1969 0 ALA 224 40. 917 30. 985 40. 490 1. 00 23. 63 BBBB ATOM 1970 N ILE 225 40. 443 31. 332 42. 667 1. 00 19. 57 BBBB ATOM 1971 H ILE 225 40. 814 31. 262 43. 573 1. 00 0. 00 BBBB ATOM 1972 CA ILE 225 39. 022 31. 595 42. 456 1. 00 18. 02 BBBB ATOM 1973 CB ILE 225 38. 139 30. 383 42. 880 1. 00 17. 65 BBBB ATOM 1974 CG2 ILE 225 38. 420 29. 200 41. 962 1. 00 18. 13 BBBB ATOM 1975 CG1 ILE 225 38. 37130. 01644. 3611. 0016. 24 BBBB ATOM 1976 CD ILE 225 37. 619 28. 785 44. 847 1. 00 14. 44 BBBB ATOM 1977 C ILE 225 38. 605 32. 846 43. 220 1. 00 19. 24 BBBB ATOM 1978 0 ILE 225 39. 239 33. 210 44. 219 1. 00 20. 04 BBBB ATOM 1979 N ALA 226 37. 665 33. 593 42. 648 1. 00 16. 66 BBBB ATOM 1980 H ALA 226 37. 258 33. 255 41. 826 1. 00 0. 00 BBBB ATOM 1981 CA ALA 226 37. 224 34. 869 43. 209 1. 00 15. 95 BBBB ATOM 1982 CB ALA 226 37. 347 35. 958 42. 154 1. 00 12. 73 BBBB ATOM 1983 C ALA 226 35. 775 34. 757 43. 679 1. 00 18. 07 BBBB ATOM 1984 0 ALA 226 34. 971 34. 086 43. 019 1. 00 18. 08 BBBB ATOM 1985 N LEU 227 35. 492 35. 237 44. 894 1. 00 18. 46 BBBB ATOM 1986 H LEU 227 36. 23535. 50145. 4491. 000. 00 BBBB ATOM 1987 CA LEU 227 34. 105 35. 292 45. 382 1. 00 18. 63 BBBB ATOM 1988 CB LEU 227 34. 034 35. 223 46. 910 1. 00 17. 78 BBBB ATOM 1989 CG LEU 227 32. 630 35. 026 47. 507 1. 00 15. 56 BBBB ATOM 1990 CD1 LEU 227 31. 934 33. 818 46. 849 1. 00 11. 24 BBBB ATOM 1991 CD2 LEU 227 32. 728 34. 824 49. 026 1. 00 16. 41 BBBB ATOM 1992 C LEU 227 33. 421 36. 564 44. 889 1. 00 18. 62 BBBB ATOM 1993 0 LEU 227 33. 644 37. 662 45. 423 1. 00 22. 19 BBBB

ATOM 1994 N SER 228 32. 617 36. 409 43. 841 1. 00 21. 18 BBBB ATOM 1995 H SER 228 32. 389 35. 506 43. 551 1. 00 0. 00 BBBB ATOM 1996 CA SER 228 32. 10137. 547 43. 082 1. 00 23. 05 BBBB ATOM 1997 CB SER 228 32. 340 37. 337 41. 583 1. 00 21. 38 BBBB ATOM 1998 OG SER 228 33. 71737. 354 41. 256 1. 00 27. 97 BBBB ATOM 1999 HG SER 228 33. 938 38. 255 40. 993 1. 00 0. 00 BBBB ATOM 2000 C SER 228 30. 61137. 740 43. 319 1. 00 22. 96 BBBB ATOM 2001 0 SER 228 29. 87936. 774 43. 537 1. 00 22. 70 BBBB ATOM 2002 N ALA 229 30. 175 38. 994 43. 240 1. 00 25. 88 BBBB ATOM 2003 H ALA 229 30. 865 39. 687 43. 183 1. 00 0. 00 BBBB ATOM 2004 CA ALA 229 28. 755 39. 339 43. 272 1. 00 27. 79 BBBB ATOM 2005 CB ALA 229 28. 57640. 816 43. 037 1. 00 26. 67 BBBB ATOM 2006 C ALA 229 27. 94738. 557 42. 253 1. 00 30. 41 BBBB ATOM 2007 0 ALA 229 28. 343 38. 421 41. 091 1. 00 28. 94 BBBB ATOM 2008 N ALA 230 26. 875 37. 943 42. 727 1. 00 34. 26 BBBB ATOM 2009 H ALA 230 26. 619 38. 098 43. 649 1. 00 0. 00 BBBB ATOM 2010 CA ALA 230 25. 93237. 264 41. 850 1. 00 39. 79 BBBB ATOM 2011 CB ALA 230 26. 227 35. 769 41. 806 1. 00 36. 23 BBBB ATOM 2012 C ALA 230 24. 515 37. 521 42. 353 1. 00 43. 03 BBBB ATOM 2013 OT1 ALA 230 24. 31738. 529 43. 082 1. 00 45. 31 BBBB ATOM 2014 OT2 ALA 230 23. 610 36. 739 41. 986 1. 00 51. 60 BBBB ATOM 2015 OH2 WAT W 1 51. 481 27. 762 54. 626 1. 00 18. 55 CCCC ATOM 2016 H1 WAT W 1 50. 686 28. 045 55. 047 1. 00 0. 00 CCCC ATOM 2017 H2 WAT W 1 51. 852 28. 563 54. 263 1. 00 0. 00 CCCC ATOM 2018 OH2 WAT W 2 39. 370 21. 410 52. 578 1. 00 15. 77 CCCC ATOM 2019 H1 WAT W 2 39. 781 20. 538 52. 650 1. 00 0. 00 CCCC ATOM 2020 H2 WAT W 2 38. 930 21. 344 51. 730 1. 00 0. 00 CCCC ATOM 2021 OH2 WAT W 3 35. 144 43. 822 54. 730 1. 00 48. 43 CCCC ATOM 2022 H1 WAT W 3 35. 076 42. 857 54. 802 1. 00 0. 00 CCSC ATOM 2023 H2 WAT W 3 36. 016 43. 867 54. 331 1. 00 0. 00 CCCC ATOM 2024 OH2 WAT W 4 39. 532 35. 665 68. 967 1. 00 39. 36 CCCC

ATOM 2025 H1 WAT W 4 38. 850 35. 846 69. 610 1. 00 0. 00 CCCC ATOM 2026 H2 WAT W 4 39. 154 34. 910 68. 494 1. 00 0. 00 CCCC ATOM 2027 OH2 WAT W 5 52. 778 35. 754 52. 431 1. 00 19. 08 CCCC ATOM 2028 H1 WAT W 5 52. 330 35. 766 53. 277 1. 00 0. 00 CCCC ATOM 2029 H2 WAT W 5 53. 715 35. 839 52. 604 1. 00 0. 00 CCCC ATOM 2030 OH2 WAT W 6 40. 635 36. 308 64. 686 1. 00 24. 09 CCCC ATOM 2031 H1 WAT W 6 40. 363 37. 194 64. 430 1. 00 0. 00 CCCC ATOM 2032 H2 WAT W 6 40. 869 36. 450 65. 613 1. 00 0. 00 CCCC ATOM 2033 OH2 WAT W 7 44. 431 27. 177 56. 600 1. 00 19. 80 CCCC ATOM 2034 H1 WAT W 7 44. 407 26. 205 56. 639 1. 00 0. 00 CCCC ATOM 2035 H2 WAT W 7 43. 481 27. 377 56. 604 1. 00 0. 00 CCCC ATOM 2036 OH2 WAT W 8 42. 402 30. 227 54. 711 1. 00 11. 84 CCCC ATOM 2037 H1 WAT W 8 43. 286 30. 482 54. 423 1. 00 0. 00 CCCC ATOM 2038 H2 WAT W 8 42. 346 29. 333 54. 345 1. 00 0. 00 CCCC ATOM 2039 OH2 WAT W 9 40. 731 28. 824 38. 939 1. 00 28. 73 CCCC ATOM 2040 H1 WAT W 9 40. 795 27. 981 38. 482 1. 00 0. 00 CCCC ATOM 2041 H2 WAT W 9 39. 802 28. 985 39. 058 1. 00 0. 00 CCCC ATOM 2042 OH2 WAT W 10 25. 085 35. 294 50. 522 1. 00 30. 46 CCCC ATOM 2043 H1 WA1 W 10 25. 848 35. 145 51. 097 1. 00 0. 00 CCCC ATOM 2044 H2 WAT W 10 25. 073 34. 478 50. 005 1. 00 0. 00 CCCC

ATOM 2045 OH2 WAT W 11 43. 212 27. 747 54. 339 1. 00 16. 77 CCCC ATOM 2046 H1 WAT W 11 42. 735 27. 129 53. 765 1. 00 0. 00 CCCC ATOM 2047 H2 WAT W 11 43. 973 27. 993 53. 794 1. 00 0. 00 CCCC ATOM 2048 OH2 WAT W 12 25. 873 28. 833 46. 097 1. 00 25. 93 CCCC ATOM 2049 H1 WAT W 12 26. 045 28. 055 46. 634 1. 00 0. 00 CCCC ATOM 2050 H2 WAT W 12 25. 143 29. 291 46. 521 1. 00 0. 00 CCCC ATOM 2051 OH2 WAT W 13 47. 247 22. 287 53. 625 1. 00 32. 05 CCCC ATOM 2052 H1 WAT W 13 47. 397 21. 925 52. 743 1. 00 0. 00 CCCC ATOM 2053 H2 WAT W 13 46. 502 21. 777 53. 977 1. 00 0. 00 CCCC ATOM 2054 OH2 WAT W 14 32. 315 33. 010 70. 814 1. 00 30. 92 CCCC ATOM 2055 H1 WAT W 14 31. 764 32. 247 70. 559 1. 00 0. 00 CCCC ATOM 2056 H2 WAT W 14 32. 482 32. 806 71. 743 1. 00 0. 00 CCCC ATOM 2057 OH2 WAT W 15 27. 439 20. 370 63. 661 1. 00 35. 31 CCCC ATOM 2058 H1 WAT W 15 27. 577 19. 523 64. 085 1. 00 0. 00 CCCC ATOM 2059 H2 WAT W 15 26. 570 20. 639 64. 000 1. 00 0. 00 CCCC ATOM 2060 OH2 WAT W 16 36. 688 34. 815 35. 452 1. 00 26. 31 CCCC ATOM 2061 H1 WAT W 16 37. 119 33. 999 35. 736 1. 00 0. 00 CCCC ATOM 2062 H2 WAT W 16 36. 741 34. 794 34. 493 1. 00 0. 00 CCCC ATOM 2063 OH2 WAT W 17 55. 000 30. 105 39. 281 1. 00 40. 98 CCCC ATOM 2064 H1 WAT W 17 55. 681 29. 609 39. 748 1. 00 0. 00 CCCC

ATOM 2065 H2 WAT W 17 54. 462 29. 407 38. 898 1. 00 0. 00 CCCC ATOM 2066 OH2 WAT W 18 43. 052 23. 709 40. 956 1. 00 40. 59 CCCC ATOM 2067 H1 WAT W 18 42. 643 24. 247 40. 269 1. 00 0. 00 CCCC ATOM 2068 H2 WAT W 18 43. 936 23. 558 40. 602 1. 00 0. 00 CCCC ATOM 2069 OH2 WAT W 19 54. 909 33. 171 58. 012 1. 00 19. 53 CCCC ATOM 2070 H1 WAT W 19 54. 343 33. 687 57. 452 1. 00 0. 00 CCCC ATOM 2071 H2 WAT W 19 55. 741 33. 098 57. 532 1. 00 0. 00 CCCC ATOM 2072 OH2 WAT W 20 40. 237 17. 628 51. 003 1. 00 33. 94 CCCC ATOM 2073 H1 WAT W 20 39. 951 18. 550 51. 077 1. 00 0. 00 CCCC ATOM 2074 H2 WAT W 20 40. 823 17. 557 51. 766 1. 00 0. 00 CCCC ATOM 2075 OH2 WAT W 21 50. 618 31. 128 52. 585 1. 00 18. 87 CCCC ATOM 2076 H1 WAT W 21 51. 070 31. 770 53. 133 1. 00 0. 00 CCCC ATOM 2077 H2 WAT W 21 49. 874 31. 595 52. 195 1. 00 0. 00 CCCC ATOM 2078 OH2 WAT W 22 47. 828 33. 973 57. 688 1. 00 12. 70 CCCC ATOM 2079 H1 WAT W 22 48. 249 33. 962 58. 551 1. 00 0. 00 CCCC ATOM 2080 H2 WAT W 22 48. 064 33. 104 57. 362 1. 00 0. 00 CCCC ATOM 2081 OH2 WAT W 23 42. 063 39. 807 64. 625 1. 00 30. 20 CCCC ATOM 2082 H1 WAT W 23 41. 289 39. 678 64. 069 1. 00 0. 00 CCCC ATOM 2083 H2 WAT W 23 42. 289 4U. 734 64. 486 1. 00 0. 00 CCCC ATOM 2084 OH2 WAT W 24 33. 765 20. 863 55. 211 1. 00 29. 13 CCCC

ATOM 2085 H1 WAT W 24 33. 127 20. 926 55. 939 1. 00 0. 00 CCCC ATOM 2086 H2 WAT W 24 33. 724 19. 909 55. 040 1. 00 0. 00 CCCC ATOM 2087 OH2 WAT W 25 36. 791 41. 478 51. 569 1. 00 21. 42 CCCC ATOM 2088 H1 WAT W 25 36. 666 42. 072 52. 329 1. 00 0. 00 CCCC ATOM 2089 H2 WAT W 25 36. 015 40. 915 51. 732 1. 00 0. 00 CCCC ATOM 2090 OH2 WAT W 26 22. 888 33. 170 41. 709 1. 00 36. 61 CCCC ATOM 2091 H1 WAT W 26 22. 945 34. 011 41. 225 1. 00 0. nO CCCC ATOM 2092 H2 WAT W 26 23. 300 33. 483 42. 535 1. 00 0. 00 CCCC ATOM 2093 OH2 WAT W 27 55. 171 39. 105 53. 741 1. 00 42. 20 CCCC ATOM 2094 H1 WAT W 27 55. 663 39. 042 54. 569 1. 00 0. 00 CCCC ATOM 2095 H2 WAT W 27 54. 806 38. 247 53. 609 1. 00 0. 00 CCCC ATOM 2096 OH2 WAT W 28 28. 321 27. 402 40. 210 1. 00 30. 51 CCCC ATOM 2097 H1 WAT W 28 28. 268 26. 785 39. 486 1. 00 0. 00 CCCC ATOM 2098 H2 WAT W 28 27. 404 27. 641 40. 409 1. 00 0. 00 CCCC ATOM 2099 OH2 WAT W 29 40. 852 16. 628 47. 914 1. 00 34. 70 CCCC ATOM 2100 H1 WAT W 29 41. 261 15. 764 48. 057 1. 00 0. 00 CCCC ATOM 2101 H2 WAT W 29 40. 593 16. 863 48. 822 1. 00 0. 00 CCCC ATOM 2102 OH2 WAT W 30 39. 338 35. 289 34. 944 1. 00 21. 05 CCCC ATOM 2103 H1 WAT W 30 38. 678 35. 160 35. 647 1. 00 0. 00 CCCC ATOM 2104 H2 WAT W 30 39. 284 36. 252 34. 809 1. 00 0. 00 CCCC

ATOM 2105 OH2 WAT W 31 33. 019 43. 572 48. 633 1. 00 37. 54 CCCC ATOM 2106 H1 WAT W 31 32. 798 44. 226 49. 297 1. 00 0. 00 CCCC ATOM 2107 H2 WAT W 31 33. 948 43. 674 48. 472 1. 00 0. 00 CCCC ATOM 2108 OH2 WAT W 32 47. 579 20. 217 52. 081 1. 00 37. 78 CCCC ATOM 2109 H1 WAT W 32 46. 890 20. 151 52. 764 1. 00 0. 00 CCCC ATOM 2110 H2 WAT W 32 47. 134 20. 719 51. 380 1. 00 0. 00 CCCC ATOM 2111 OH2 WAT W 33 35. 901 43. 755 50. 596 1. 00 26. 42 CCCC ATOM 2112 H1 WAT W 33 35. 376 44. 062 49. 857 1. 00 0. 00 CCCC ATOM 2113 H2 WAT W 33 36. 097 42. 838 50. 344 1. 00 0. 00 CCCC ATOM 2114 OH2 WAT W 34 42. 104 46. 370 66. 356 1. 00 30. 52 CCCC ATOM 2115 H1 WAT W 34 41. 961 46. 330 67. 306 1. 00 0. 00 CCCC ATOM 2116 H2 WAT W 34 42. 851 46. 966 66. 269 1. 00 0. 00 CCCC ATOM 2117 OH2 WAT W 35 46. 131 17. 969 52. 630 1. 00 35. 74 CCCC ATOM 2118 H1 WAT W 35 45. 557 17. 546 51. 952 1. 00 0. 00 CCCC ATOM 2119 H2 WAT W 35 46. 805 18. 440 52. 129 1. 00 0. 00 CCCC ATOM 2120 OH2 WAT W 36 52. 356 28. 587 62. 695 1. 00 31. 73 CCCC ATOM 2121 H1 WAT W 36 52. 236 28. 111 61. 849 1. 00 0. 00 CCCC ATOM 2122 H2 WAT W 36 53. 107 28. 128 63. 068 1. 00 0. 00 CCCC ATOM 2123 OH2 WAr W 37 53. 928 32. 954 52. 385 1. 00 34. 97 CCCC ATOM 2124 H1 WAT W 37 54. 027 33. 054 51. 433 1. 00 0. 00 CCCC

ATOM 2125 H2 WAT W 37 54. 168 33. 807 52. 721 1. 00 0. 00 CCCC ATOM 2126 OH2 WAT W 38 54. 246 30. 819 59. 312 1. 00 24. 90 CCCC ATOM 2127 H1 WAT W 38 54. 899 31. 330 58. 817 1. 00 0. 00 CCCC ATOM 2128 H2 WAT W 38 54. 625 30. 713 60. 178 1. 00 0. 00 CCCC ATOM 2129 OH2 WAT W 39 52. 917 41. 940 57. 637 1. 00 31. 49 CCCC ATOM 2130 H1 WAT W 39 52. 509 42. 286 56. 832 1. 00 0. 00 CCCC ATOM 2131 H2 WAT W 39 52. 166 41. 650 58. 142 1. 00 0. 00 CCCC ATOM 2132 OH2 WAT W 40 22. 703 25. 095 56. 301 1. 00 26. 37 CCCC ATOM 2133 H1 WAT W 40 22. 608 24. 325 55. 722 1. 00 0. 00 CCCC ATOM 2134 H2 WAT W 40 21. 849 25. 138 56. 770 1. 00 0. 00 CCCC ATOM 2135 OH2 WAT W 41 30. 795 30. 259 38. 753 1. 00 31. 81 CCCC ATOM 2136 H1 WAT W 41 31. 181 30. 659 39. 538 1. 00 0. 00 CCCC ATOM 2137 H2 WAT W 41 30. 536 30. 996 38. 213 1. 00 0. 00 CCCC ATOM 2138 OH2 WAT W 42 27. 059 32. 642 48. 470 1. 00 29. 12 CCCC ATOM 2139 H1 WAT W 42 26. 179 32. 491 48. 826 1. 00 0. 00 CCCC ATOM 2140 H2 WAT W 42 26. 887 32. 882 47. 561 1. 00 0. 00 CCCC ATOM 2141 OH2 WAT W 43 31. 741 35. 155 63. 468 1. 00 34. 61 CCCC ATOM 2142 H1 WAT W 43 32. 424 35. 645 62. 988 1. 00 0. 00 CCCC ATOM 2143 H2 WAT W 43 32. 162 34. 352 63. 777 1. 00 0. 00 CCCC ATOM 2144 OH2 WAT W 44 29. 727 30. 243 71. 731 1. 00 31. 51 CCCC

ATOM 2145 H1 WAT W 44 28. 793 30. 072 71. 838 1. 00 0. 00 CCCC ATOM 2146 H2 WAT W 44 29. 847 30. 411 70. 803 1. 00 0. 00 CCCC ATOM 2147 OH2 WAT W 45 36. 050 33. 785 74. 656 1. 00 35. 20 CCCC ATOM 2148 H1 WAT W 45 36. 659 33. 064 74. 675 1. 00 0. 00 CCCC ATOM 2149 H2 WAT W 45 36. 197 34. 091 73. 746 1. 00 0. 00 CCCC ATOM 2150 OH2 WAT W 46 39. 267 26. 175 75. 132 1. 00 43. 10 CCCC ATOM 2151 H1 WAT W 46 38. 838 26. 792 74. 559 1. 00 0. 00 CCCC ATOM 2152 H2 WAT W 46 38. 723 26. 196 75. 918 1. 00 0. 00 CCCC ATOM 2153 OH2 WAT W 47 54. 676 27. 697 46. 296 1. 00 31. 80 CCCC ATOM 2154 H1 WAT W 47 54. 334 28. 563 46. 038 1. 00 0. 00 CCCC ATOM 2155 H2 WAT W 47 55. 478 27. 593 45. 804 1. 00 0. 00 CCCC ATOM 2156 OH2 WAT W 48 43. 162 29. 556 72. 942 1. 00 30. 66 CCCC ATOM 2157 H1 WAT W 48 42. 388 29. 317 73. 466 1. 00 0. 00 CCCC ATOM 2158 H2 WAT W 48 43. 883 29. 297 73. 531 1. 00 0. 00 CCCC ATOM 2159 OH2 WAT W 49 21. 891 36. 820 56. 313 1. 00 47. 38 CCCC ATOM 2160 H1 WAT W 49 22. 570 37. 512 56. 485 1. 00 0. 00 CCCC ATOM 2161 H2 WAT W 49 21. 504 36. 758 57. 185 1. 00 0. 00 CCCC ATOM 2162 OH2 WAT W 50 21. 789 29. 233 61. 759 1. 00 31. 36 CCCC ATOM 2163 H1 WAT W 50 22. 579 28. 751 61. 504 1. 00 0. 00 CCCC ATOM 2164 H2 WAT W 50 21. 888 29. 397 62. 696 1. 00 0. 00 CCCC

ATOM 2165 OH2 WAT W 51 24. 553 38. 700 56. 934 1. 00 33. 19 CCCC ATOM 2166 H1 WAT W 51 25. 457 38. 781 57. 260 1. 00 0. 00 CCCC ATOM 2167 H2 WAT W 51 24. 725 38. 492 56. 006 1. 00 0. 00 CCCC ATOM 2168 OH2 WAT W 52 37. 071 21. 855 45. 561 1. 00 31. 91 CCCC ATOM 2169 H1 WAT W 52 37. 534 21. 483 46. 320 1. 00 0. 00 CCCC ATOM 2170 H2 WAT W 52 36. 195 21. 465 45. 599 1. 00 0. 00 CCCC ATOM 2171 OH2 WAT W 53 36. 340 41. 156 54. 064 1. 00 35. 66 CCCC ATOM 2172 H1 WAT W 53 36. 279 41. 345 55. 006 1. 00 0. 00 CCCC ATOM 2173 H2 WAT W 53 35. 608 40. 534 53. 971 1. 00 0. 00 CCCC ATOM 2174 OH2 WAT W 54 38. 081 43. 889 43. 261 1. 00 36. 97 CCCC ATOM 2175 H1 WAT W 54 37. 336 43. 702 43. 845 1. 00 0. 00 CCCC ATOM 2176 H2 WAT W 54 37. 896 44. 806 43. 010 1. 00 0. 00 CCCC ATOM 2177 OH2 WAT W 55 35. 518 44. 375 43. 828 1. 00 32. 62 CCCC ATOM 2178 H1 WAT W 55 35. 620 44. 696 44. 737 1. 00 0. 00 CCCC ATOM 2179 H2 WAT W 55 35. 338 45. 220 43. 378 1. 00 0. 00 CCCC ATOM 2180 OH2 WAT W 56 36. 543 49. 730 50. 862 1. 00 38. 31 CCCC ATOM 2181 H1 WAT W 56 35. 701 49. 253 50. 861 1. 00 0. 00 CCCC ATOM 2182 H2 WAT W 56 36. 221 50. 651 50. 872 1. 00 0. 00 CCCC ATOM 2183 OH2 WAT W 57 47. 923 21. 229 58. 848 1. 00 36. 53 CCCC ATOM 2184 H1 WAT W 57 47. 602 21. 346 59. 752 1. 00 0. 00 CCCC

ATOM 2185 H2 WAT W 57 47. 861 20. 309 58. 628 1. 00 0. 00 CCCC ATOM 2186 OH2 WAT W 58 50. 570 39. 535 64. 284 1. 00 25. 23 CCCC ATOM 2187 H1 WAT W 58 50. 813 40. 161 64. 972 1. 00 0. 00 CCCC ATOM 2188 H2 WAT W 58 50. 606 38. 686 64. 732 1. 00 0. 00 CCCC ATOM 2189 OH2 WAT W 59 37. 290 16. 000 50. 508 1. 00 40. 28 CCCC ATOM 2190 H1 WAT W 59 38. 133 15. 923 50. 048 1. 00 0. 00 CCCC ATOM 2191 H2 WAT W 59 37. 169 16. 957 50. 545 1. 00 0. 00 CCCC ATOM 2192 OH2 WAT W 60 42. 786 46. 386 58. 648 1. 00 42. 06 CCCC ATOM 2193 H1 WAT W 60 42. 214 47. 140 58. 450 1. 00 0. 00 CCCC ATOM 2194 H2 WAT W 60 42. 290 45. 678 58. 198 1. 00 0. 00 CCCC ATOM 2195 OH2 WAT W 61 45. 484 16. 421 56. 551 1. 00 31. 57 CCCC ATOM 2196 H1 WAT W 61 45. 506 16. 256 55. 607 1. 00 0. 00 CCCC ATOM 2197 H2 WAT W 61 46. 388 16. 730 56. 637 1. 00 0. 00 CCCC ATOM 2198 OH2 WAT W 62 31. 187 16. 050 67. 214 1. 00 45. 70 CCCC ATOM 2199 H1 WAT W 62 31. 924 16. 489 67. 675 1. 00 0. 00 CCCC ATOM 2200 H2 WAT W 62 31. 297 16. 473 66. 357 1. 00 0. 00 CCCC ATOM 2201 OH2 WAT W 63 25. 531 30. 675 47. 998 1. 00 31. 29 CCCC ATOM 2202 H1 WAT W 63 25. 137 30. 008 48. 585 1. 00 0. 00 CCCC ATOM 2203 H2 WAT W 63 26. 353 30. 264 47. 682 1. 00 0. 00 CCCC ATOM 2204 OH2 WAT W 64 34. 733 30. 790 35. 948 1. 00 30. 50 CCCC

ATOM 2205 H1 WAT W 64 35. 036 29. 917 36. 213 1. 00 0. 00 CCCC ATOM 2206 H2 WAT W 64 34. 052 31. 003 36. 592 1. 00 0. 00 CCCC ATOM 2207 OH2 WAT W 65 35. 632 43. 962 48. 122 1. 00 39. 10 CCCC ATOM 2208 H1 WAT W 65 35. 906 44. 347 47. 268 1. 00 0. 00 CCCC ATOM 2209 H2 WAT W 65 35. 638 43. 021 47. 836 1. 00 0. 00 CCCC ATOM 2210 OH2 WAT W 66 41. 405 29. 179 35. 736 1. 00 55. 14 CCCC ATOM 2211 H1 WAT W 66 40. 685 28. 791 36. 221 1. 00 0. 00 CCCC ATOM 2212 H2 WAT W 66 41. 628 29. 970 36. 248 1. 00 0. 00 CCCC ATOM 2213 OH2 WAT W 67 47. 648 45. 349 48. 932 1. 00 33. 82 CCCC ATOM 2214 H1 WAT W 67 47. 167 46. 150 49. 188 1. 00 0. 00 CCCC ATOM 2215 H2 WAT W 67 47. 436 45. 253 47. 995 1. 00 0. 00 CCCC ATOM 2216 OH2 WAT W 68 44. 618 17. 410 45. 691 1. 00 53. 27 CCCC ATOM 2217 H1 WAT W 68 43. 969 16. 949 45. 154 1. 00 0. 00 CCCC ATOM 2218 H2 WAT W 68 44. 364 18. 336 45. 569 1. 00 0. 00 CCCC ATOM 2219 OH2 WAT W 69 27. 329 25. 288 41. 675 1. 00 38. 34 CCCC ATOM 2220 H1 WAT W 69 27. 449 25. 214 40. 731 1. 00 0. 00 CCCC ATOM 2221 H2 WAT W 69 28. 180 25. 002 42. 032 1. 00 0. 00 CCCC ATOM 2222 OH2 WAT W 70 27. 444 23. 990 68. 137 1. 00 30. 31 CCCC ATOM 2223 H1 WAT W 7U 27. 833 24. 141 69. 000 1. 00 0. 00 CCCC ATOM 2224 H2 WAT W 70 26. 670 23. 444 68. 324 1. 00 0. 00 CCCC

ATOM 2225 OH2 WAT W 71 48. 599 44. 086 46. 923 1. 00 27. 72 CCCC ATOM 2226 H1 WAT W 71 49. 053 44. 453 47. 702 1. 00 0. 00 CCCC ATOM 2227 H2 WAT W 71 48. 172 43. 287 47. 257 1. 00 0. 00 CCCC ATOM 2228 OH2 WAT W 72 36. 454 19. 649 44. 328 1. 00 44. 87 CCCC ATOM 2229 H1 WAT W 72 37. 251 19. 129 44. 245 1. 00 0. 00 CCCC ATOM 2230 H2 WAT W 72 36. 454 20. 184 43. 535 1. 00 0. 00 CCCC ATOM 2231 OH2 WAT W 73 32. 924 36. 639 61. 406 1. 00 49. 32 CCCC ATOM 2232 H1 WAT W 73 32. 799 36. 561 60. 455 1. 00 0. 00 CCCC ATOM 2233 H2 WAT W 73 33. 399 37. 476 61. 457 1. 00 0. 00 CCCC ATOM 2234 OH2 WAT W 74 34. 350 46. 549 42. 555 1. 00 37. 93 CCCC ATOM 2235 H1 WAT W 74 34. 920 46. 905 41. 855 1. 00 0. 00 CCCC ATOM 2236 H2 WAT W 74 33. 481 46. 561 42. 137 1. 00 0. 00 CCCC ATOM 2237 OH2 WAT W 75 41. 866 22. 222 61. 197 1. 00 23. 90 CCCC ATOM 2238 H1 WAT W 75 41. 267 21. 986 60. 474 1. 00 0. 00 CCCC ATOM 2239 H2 WAT W 75 41. 725 23. 181 61. 165 1. 00 0. 00 CCCC ATOM 2240 OH2 WAT W 76 40. 603 24. 476 61. 528 1. 00 26. 60 CCCC ATOM 2241 H1 WAT W 76 40. 035 24. 049 62. 178 1. 00 0. 00 CCCC ATOM 2242 H2 WAT W 76 41. 129 25. 098 62. 053 1. 00 0. 00 CCCC ATOM 2243 OH2 WAT W 77 37. 587 42. 214 37. 006 1. 00 27. 49 CCCC ATOM 2244 H1 WAT W 77 36. 716 41. 799 37. 094 1. 00 0. 00 CCCC

ATOM 2245 H2 WAT W 77 38. 120 41. 519 36. 628 1. 00 0. 00 CCCC ATOM 2246 OH2 WAT W 78 37. 491 37. 774 65. 510 1. 00 33. 68 CCCC ATOM 2247 H1 WAT W 78 37. 311 38. 514 66. 088 1. 00 0. 00 CCCC ATOM 2248 H2 WAT W 78 38. 196 38. 100 64. 942 1. 00 0. 00 CCCC ATOM 2249 OH2 WAT W 79 30. 893 42. 450 55. 672 1. 00 37. 91 CCCC ATOM 2250 H1 WAT W 79 31. 383 43. 287 55. 656 1. 00 0. 00 CCCC ATOM 2251 H2 WAT W 79 30. 446 42. 490 56. 512 1. 00 0. 00 CCCC ATOM 2252 OH2 WAT W 80 41. 590 26. 364 75. 819 1. 00 44. 98 CCCC ATOM 2253 H1 WAT W 80 40. 945 26. 762 75. 228 1. 00 0. 00 CCCC ATOM 2254 H2 WAT W 80 40. 977 25. 829 76. 331 1. 00 0. 00 CCCC ATOM 2255 OH2 WAT W 81 31. 551 16. 644 69. 763 1. 00 40. 28 CCCC ATOM 2256 H1 WAT W 81 30. 876 16. 454 69. 097 1. 00 0. 00 CCCC ATOM 2257 H2 WAT W 81 31. 289 16. 065 70. 482 1. 00 0. 00 CCCC ATOM 2258 OH2 WAT W 82 37. 407 25. 467 44. 875 1. 00 41. 83 CCCC ATOM 2259 H1 WAT W 82 38. 074 24. 748 44. 931 1. 00 0. 00 CCCC ATOM 2260 H2 WAT W 82 36. 624 24. 959 45. 163 1. 00 0. 00 CCCC ATOM 2261 OH2 WAT W 83 36. 050 44. 143 66. 742 1. 00 47. 55 CCCC ATOM 2262 H1 WAT W 83 35. 853 45. 034 67. 047 1. 00 0. 00 CCCC ATOM 2263 H2 WAT W 83 35. 202 43. 808 66. 440 1. 00 0. 00 CCCC ATOM 2264 OH2 WAT W 84 55. 079 30. 486 46. 914 1. 00 33. 99 CCCC

ATOM 2265 H1 WAT W 84 55. 533 29. 662 47. 105 1. 00 0. 00 CCCC ATOM 2266 H2 WAT W 84 55. 427 30. 802 46. 067 1. 00 0. 00 CCCC ATOM 2267 OH2 WAT W 85 19. 795 23. 892 60. 011 1. 00 45. 91 CCCC ATOM 2268 H1 WAT W 85 20. 565 24. 394 60. 299 1. 00 0. 00 CCCC ATOM 2269 H2 WAT W 85 19. 513 24. 429 59. 269 1. 00 0. 00 CCCC ATOM 2270 OH2 WAT W 86 25. 526 25. 281 69. 586 1. 00 50. 15 CCCC ATOM 2271 H1 WAT W 86 26. 156 25. 419 68. 861 1. 00 0. 00 CCCC ATOM 2272 H2 WAT W 86 25. 078 26. 130 69. 601 1. 00 0. 00 CCCC ATOM 2273 OH2 WAT W 87 33. 162 15. 779 64. 852 1. 00 52. 39 CCCC ATOM 2274 H1 WAT W 87 32. 401 16. 289 64. 587 1. 00 0. 00 CCCC ATOM 2275 H2 WAT W 87 32. 842 14. 948 65. 194 1. 00 0. 00 CCCC ATOM 2276 OH2 WAT W 88 51. 078 46. 719 59. 813 1. 00 45. 28 CCCC ATOM 2277 H1 WAT W 88 51. 110 47. 176 58. 964 1. 00 0. 00 CCCC ATOM 2278 H2 WAT W 88 50. 807 47. 444 60. 384 1. 00 0. 00 CCCC ATOM 2279 OH2 WAT W 89 29. 000 40. 539 39. 389 1. 00 37. 09 CCCC ATOM 2280 H1 WAT W 89 28. 599 39. 760 39. 809 1. 00 0. 00 CCCC ATOM 2281 H2 WAT W 89 29. 294 41. 057 40. 144 1. 00 0. 00 CCCC ATOM 2282 OH2 WAT W 90 47. 032 24. 721 42. 183 1. 00 36. 90 CCCC ATOM 2283 H1 WAT W 90 46. 128 24. 591 42. 465 1. 00 0. 00 CCCC ATOM 2284 H2 WAT W 90 47. 379 25. 426 42. 760 1. 00 0. 00 CCCC

ATOM 2285 OH2 WAT W 91 44. 324 30. 773 38. 366 1. 00 57. 11 CCCC ATOM 2286 H1 WAT W 91 44. 569 30. 042 37. 778 1. 00 0. 00 CCCC ATOM 2287 H2 WAT W 91 43. 570 31. 159 37. 888 1. 00 0. 00 CCCC ATOM 2288 OH2 WAT W 92 49. 852 49. 467 51. 199 1. 00 42. 65 CCCC ATOM 2289 H1 WAT W 92 49. 178 50. 143 51. 105 1. 00 0. 00 CCCC ATOM 2290 H2 WAT W 92 49. 580 48. 789 50. 564 1. 00 0. 00 CCCC ATOM 2291 OH2 WAT W 93 34. 519 38. 964 70. 014 1. 00 48. 33 CCCC ATOM 2292 H1 WAT W 93 34. 947 39. 776 69. 730 1. 00 0. 00 CCCC ATOM 2293 H2 WAT W 93 34. 689 38. 364 69. 262 1. 00 0. 00 CCCC ATOM 2294 OH2 WAT W 94 42. 967 50. 912 67. 215 1. 00 39. 91 CCCC ATOM 2295 H1 WAT W 94 43. 243 51. 292 66. 379 1. 00 0. 00 CCCC ATOM 2296 H2 WAT W 94 43. 165 51. 598 67. 856 1. 00 0. 00 CCCC ATOM 2297 OH2 WAT W 95 47. 936 18. 190 48. 416 1. 00 51. 10 CCCC ATOM 2298 H1 WAT W 95 47. 199 18. 806 48. 599 1. 00 0. 00 CCCC ATOM 2299 H2 WAT W 95 47. 397 17. 492 47. 973 1. 00 0. 00 CCCC ATOM 2300 OH2 WAT W 96 54. 886 28. 124 62. 758 1. 00 43. 49 CCCC ATOM 2301 H1 WAT W 96 55. 089 28. 298 63. 676 1. 00 0. 00 CCCC ATOM 2302 H2 WAT W 96 55. 256 28. 857 62. 269 1. 00 0. 00 CCCC ATOM 2303 OH2 WAT W 97 29. 053 34. 319 38. 220 1. 00 39. 58 CCCC ATOM 2304 H1 WAT W 97 28. 737 33. 944 37. 383 1. 00 0. 00 CCCC

ATOM 2305 H2 WAT W 97 29. 432 33. 555 38. 594 1. 00 0. 00 CCCC ATOM 2306 OH2 WAT W 98 29. 173 36. 014 40. 224 1. 00 41. 00 CCCC ATOM 2307 H1 WAT W 98 28. 640 36. 738 39. 874 1. 00 0. 00 CCCC ATOM 2308 H2 WAT W 98 29. 292 35. 366 39. 527 1. 00 0. 00 CCCC ATOM 2309 OH2 WAT W 99 51. 721 42. 553 47. 389 1. 00 41. 76 CCCC ATOM 2310 H1 WAT W 99 51. 081 42. 890 48. 009 1. 00 0. 00 CCCC ATOM 2311 H2 WAT W 99 51. 195 42. 024 46. 779 1. 00 0. 00 CCCC ATOM 2312 OH2 WAT W 100 20. 643 35. 624 58. 943 1. 00 43. 37 CCCC ATOM 2313 H1 WAT W 100 20. 409 35. 144 58. 139 1. 00 0. 00 CCCC ATOM 2314 H2 WAT W 100 20. 545 36. 550 58. 745 1. 00 0. 00 CCCC ATOM 2315 OH2 WAT W 101 20. 439 26. 085 55. 577 1. 00 39. 03 CCCC ATOM 2316 H1 WAT W 101 19. 883 26. 619 55. 020 1. 00 0. 00 CCCC ATOM 2317 H2 WAT W 101 20. 888 25. 519 54. 938 1. 00 0. 00 CCCC ATOM 2318 OH2 WAT W 102 59. 643 24. 389 43. 849 1. 00 39. 53 CCCC ATOM 2319 H1 WAT W 102 58. 961 24. 708 44. 432 1. 00 0. 00 CCCC ATOM 2320 H2 WAT W 102 59. 793 25. 086 43. 222 1. 00 0. 00 CCCC ATOM 2321 OH2 WAT W 103 60. 566 27. 542 41. 229 1. 00 46. 40 CCCC ATOM 2322 H1 WAT W 103 59. 682 27. 689 41. 545 1. 00 0. 00 CCCC ATOM 2323 H2 WAT W 103 60. 987 27. 015 41. 915 1. 00 0. 00 CCCC ATOM 2324 OH2 WAT W 104 15. 551 30. 295 55. 867 1. 00 40. 24 CCCC

ATOM 2325 H1 WAT W 104 14. 606 30. 439 55. 676 1. 00 0. 00 CCCC ATOM 2326 H2 WAT W 104 15. 858 29. 940 55. 028 1. 00 0. 00 CCCC END The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Structure Determination Crystals of native and selenomethione-substituted UCH-L3 were grown in space group P2, 2, 21 (a=48. 6 A, b=60. 8 A, c=81. 4 A). There is one molecule in the asymmetric unit and the solvent content is 48%. The structure of selenomethione- substituted UCH-L3 was determined at 2. 35 A resolution by the method of multiwavelength anomalous dispersion (MAD) (FIG. 2). The native structure was subsequently refined against 1. 8 A data to an Rvalue of 23. 0% (free Rvalue = 28. 6%) with good stereochemistry (RMSD bonds = 0. 010 A). The current refined UCH-L3 model contains 205 of the 230 residues. Three regions of UCH-L3 lack defined electron density and have been omitted from the model (residues 1-4, 147-166 and 218). The side chains of Arg-145 and Glu-203 also lack defined density and have been included in the model with occupancy of zero.

Structure of UCH-L3 UCH-L3 has overall dimensions of 43 A x 32 A x 37 A. The structure is organized around a central six-stranded antiparallel P-sheet and two long a-helices.

His-169 and Asp-184, which have both been implicated in catalysis, are located at the amino and carboxyl-terminal ends of strands 3 and strand 4 respectively. The right lobe includes a long buried a-helix (helix 4) which contains the active site nucleophile Cys-95, and a cluster of smaller helices. Helix 4 makes predominantly hydrophobic interactions with the (3-sheet, several helices, and an extended segment. The active site of UCH-L3 is located between the molecule's two lobes, within a long cleft that appears to be closed in this unliganded structure. As discussed below, the catalytic nucleophile Cys-95, the general base His-169, and Asp-184, form a catalytic triad that, along with other structural features, resembles the well known family of papain- like cysteine proteases (see FIG. 4).

A predicted secondary structure assignment was recently proposed for UCH- L3 and other UCH isozymes (Larsen et al., 1996) using the neural network program of the PredictProtein server (Rost and Sander, 1993). This analysis predicted 34% a- helical content and 17% (3-sheet for UCH-L3, which is similar to the observation of 37% a-helix and 20% (3-sheet in the crystal structure. The PredictProtein server correctly predicted 5 out of 7 helices, and 3 out of 6 strands. However, a number of important secondary structural elements in the crystal structure are misidentified by the prediction, including helix 4, which contains the active site nucleophile, Cys-95, and strand 4, which terminates one residue before Asp-184, the third member of the catalytic triad.

Comparison with Other Structures Although several well characterized classes of enzymes are known to have active site triads that apparently function to orient and activate either cysteine or serine nucleophiles, comparisons show that the papain family of cysteine proteases (Rawlings and Barrett, 1994), has great similarity with UCH-L3. The present inventors compared 21 papain-like structures that have been deposited in the Brookhaven database to UCH-L3 (FIG. 4 and FIG. 5). Of the papain-like structures, 3 are free enzyme, 4 have the active Cys bound either to oxygen atoms, 2-

mercaptoethanol or metal ion, and 14 are inhibitor complexes ; 13 are of papain, 4 cathepsin B, 3 actinidin, and 1 glycyl endopeptidase. Of the papain-like enzymes, cathepsin B has the structure with greatest overall similarity to UCH-L3 as indicated by a search performed with the Dali algorithm (Holm and Sander, 1993).

Overlap of the UCH-L3 active site triad (Cys-95, His-169, Asp-184) with the active site Cys, His, and Asn of the papain-like enzymes yields RMSD values on the three Ca atoms of between 0. 07 A and 0. 32 A for 21 papain-like structures in the Brookhaven protein data base (FIG. 4). In addition, UCH-L3 Gln-89 is structurally equivalent to Gln-19 of papain, which participates in the formation of a catalytically important structure known as the oxyanion hole (Drenth et al., 1976 ; Ménard et al., 1991 ; Schröder et al., 1993). Overlap of all four of these UCH-L3 active site residues on the papain-like enzymes yields RMSD values that range from 0. 59 A to 0. 79 A for C'atoms, and from 0. 84 A to 1. 2 A for all atoms. Interestingly, the structural similarity extends to three buried water molecules of UCH-L3 that are located between the two lobes of the protein below the active site Cys and His. Two of these water molecules are also found in the papain-like enzymes, with the third site occupied by a serine side chain. It is possible that these conserved water molecules serve architectural roles to allow juxtaposition of the two lobes of the enzyme. It is also possible that they function in catalysis, either by facilitating conformational change (Rashin et al., 1986) or substrate binding (Meyer et al., 1988).

Structural similarity at the active sites suggests that the catalytic mechanism of UCHs will resemble that of the papain-like enzymes (Storer and Menard, 1994).

Thus, it is likely that UCH-L3 Cys-95 and His-169 form a thiolate/imidazolium ion pair, Asp-184 functions to orient the enzyme active site and perhaps to stabilize the protonated form of His-169, and Gln-89 contributes to the oxyanion hole. These roles in catalysis are consistent with mutagenesis data for the Cys, His, and Asp residues of UCH-L 1 (Larsen et al., 1996). In the unliganded structure, it appears unlikely that the Cys-95 side chain is deprotonated because the carbonyl oxygen atom of Ser-92 is

positioned to form a linear 3. 2 A hydrogen bond with the Cys-95 thiol. It is likely that the thiolate ion will form after displacement of Ser-92, which, as discussed below, is expected to undergo conformational change upon substrate binding.

Starting from overlap on the active-site tetrad Ca atoms, optimal Ca superpositions of UCH-L3 with the papain-like enzymes were obtained using the program LSQMAN (Kleywegt and Jones, 1994). The best overlays were obtained with cathepsin B (Turk et al., 1995) which shows 53 equivalent Ca atoms with a RMSD of 1. 6 A. The second best agreement is found with papain (Kamphuis et al., 1984), which shows 39 equivalent C'atoms and an RMSD of 1. 2 A. Superposition of UCH-L3 with papain on the 53 Ca atoms of the optimal UCH-L3/cathepsin B overlap resulted in an RMSD of 2. 45 A.

Segments of UCH-L3 that have structural equivalents in papain-like enzymes include most of the central antiparallel (3-sheet, helix 4 (which contains the active site Cys), and an extended (3-like segment adjacent to helix 4 (FIG. 5). The major difference between these structures is that the active site helix precedes the P-sheet in papain, while the active site helix is formed from the sequence following the second 5-strand of the sheet in UCH-L3. This may have important functional consequences because it allows the positioning of a disordered loop of 20 residues over the active site of UCH-L3. As discussed below, this loop may play a role in substrate selection by the UCH enzymes.

A likely mode of substrate binding to UCH-L3 is suggested by analogy with complexes of papain-like enzymes, in which bound inhibitors occupy either the S or S'sites (FIG. 6). (Substrate residues amino-and carboxyl-terminal to the scissile bond are designated P and P'respectively, and the corresponding binding sites on the enzyme designated S and S') (Schechter and Berger, 1967). The corresponding putative active-site cleft of UCH-L3 is closed by two short segments of the enzyme, which as described below, suggests a location to allow substrate binding. This

proposed location for the UCH active site cleft is supported by the clustering of invariant surface-exposed residues in the region of the S site inhibitors of papain-like enzymes (FIG. 6C). This pattern of conserved residues is consistent with the very high specificity of UCH enzymes for ubiquitin, which is expected to bind to the proposed S sites, and the lack of selection for residues following ubiquitin, which are expected to bind in the proposed S'sites.

Further insight on substrate binding is provided by the observation that UCH- L3 binds to ubiquitin with a micromolar dissociation constant and that this interaction has a significant electrostatic component (Larsen et al., 1996). It is likely that the positively charged basic face of ubiquitin (Wilkinson, 1988) will bind to UCH enzymes. Consistent with this idea, UCH-L3 has a molecular surface of almost entirely negative electrostatic potential (Nicholls et al., 1991), including three invariant carboxylates (Glu-10, Glu-14, and Asp-33) at the putative S sites. As shown in FIG. 7, the present invention shows crudely docked ubiquitin against the proposed S sites of UCH-L3 so that electrostatic interactions appear favorable and the flexible C-terminal residues of ubiquitin are positioned analogously to the S site inhibitor of papain-like enzymes, with the ubiquitin C-terminus adjacent to the active site nucleophile, Cys-95. Hydrophobic surfaces on ubiquitin and UCH-L3 are also likely to contribute to the binding interaction.

Substrate Induced Conformational Changes Comparison with ligand-bound complexes of papain-like enzymes suggests that the specificity of UCH enzymes for ubiquitin adducts may result, in part, from maintenance of an inactive enzyme conformation in the absence of a bound ubiquitin moiety. In the absence of a binding partner, the UCH-L3 active-site cleft appears to be closed by two loops (FIG. 8). The first of these loops includes Leu-9 and Glu-10, which are in van der Waals contact with groups on the opposite side of the cleft, and are in positions incompatible with the placement of papain-like enzyme inhibitors after least squares overlap on active site residues. It also seems likely that residues 11

and 12 will have to move in order to accommodate substrate. Interestingly, Glu-10 is one of the few surface exposed UCH residues that is invariant, and it is possible that binding of positively charged groups on ubiquitin to Glu-10 initiates opening of the UCH active site cleft.

The second loop that appears to block the active site, residues 90-94, spans the catalytic residues Gln-89 and Cys-95, and adopts a conformation that differs from the equivalent region of papain-like structures by displacements of more that 4 A for the C'atoms of residues 92 and 93. Consequently, the carbonyl oxygen of UCH-L3 Ser- 92 is buried into the oxyanion hole in a position analogous to the oxygen atom of inhibitors seen in the cysteine protease inhibitor/complex structures. The Ser-92 hydroxyl forms hydrogen bonding interactions with both the thiol and main chain amide of Cys-95. Because the adjacent residue, Asn-93, is both highly exposed and invariant, it is likely that this side chain may participate in substrate binding, thereby providing a mechanism to open the active site. Conformational change in both of the loops that appear to block the active site may be coupled since van der Waals contacts are observed from residue 9 to 93 and from 6 to 93 and 94.

Access to the active site appears to be further restricted by a 20 residue disordered loop consisting of residues 147 to 166 which spans the active site cleft.

This loop may exist in several different conformations, and as discussed below, it is likely that it functions in the definition of substrate specificity. The observation of van der Waals contact between residues 7 and 146, and a hydrogen bonding interaction between residues 5 and 146 in the UCH-L3 crystal structure suggests the possibility of a coordinated conformational change upon substrate binding that includes the disordered loop.

Masking of the UCH active site in the absence of bound substrate may function to limit non-specific cleavages by these cytoplasmic proteases. An analogous conformational change probably does not occur for the papain-like

enzymes. Inspection of the liganded and unliganded structures in the Brookhaven database shows no significant conformational changes in the enzyme S sites upon binding inhibitor. The papain-like enzymes, which are generally secreted or lysosomal, employ an alternative strategy to limit inappropriate reactions. Inhibitory N-terminal propeptide extensions are cleaved only after import into the lysosome (Carmona et al., 1996 ; Coulombe et al., 1996 ; Cygler et al., 1996 ; Karrer et al., 1993 ; Turk et al., 1996).

Substrate Specificity Although UCH-L3 has high specificity for ubiquitin N-terminal to the scissile bond, it is permissive for the residues following ubiquitin provided the adduct is small and unstructured. One possible rationale for the lack of activity against larger folded C-terminal ubiquitin fusions is that only highly extended substrates can be accommodated in a deep narrow groove of UCH S'sites. The UCH-L3 crystal structure does not appear to possess such a groove, however, and thus the ordered protein visible in the crystal structure does not obviously explain the preference of UCH enzymes for small unfolded substrates. Although it is possible that a deep S' site substrate cleft could be formed by conformation change upon binding to a substrate, the very low discrimination shown across a broad rage of sequences that are cleaved from the ubiquitin C-terminus argues against this possibility.

Specific UCH Active Site Modifications More subtle modifications and changes may be made in the structure of the encoded UCH-L3 polypeptides of the present invention and still obtain a molecule that encodes a protein or peptide with characteristics of the natural UCH-L3 polypeptides, including the variants described above. The following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. The amino acid changes may be achieved by changing the codons of the DNA sequence, according to the following codon table, Table A : Table A Amino Acid Names and Codons Abbreviations Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG

Table A (continued) Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine Leu L WA WG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GW Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU It is known that certain amino acids may be substituted for other amino acids in a protein structure in order to modify or improve its antigenicity or activity (e. g., Kyte and Doolittle, 1982 ; Hopp, U. S. Patent 4, 554, 101). For example, through the substitution of alternative amino acids, small conformational changes may be conferred upon a polypeptide which result in increased activity or stability.

Alternatively, amino acid substitutions in certain polypeptides may be utilized to provide residues which may then be linked to other molecules to provide peptide- molecule conjugates which retain enough antigenicity of the starting peptide to be useful for other purposes. For example, a selected UCH-L3 peptide bound to a solid support might be constructed which would have particular advantages in diagnostic embodiments.

The importance of the hydropathic index of amino acids in conferring interactive biological function on a protein has been discussed generally by Kyte and Doolittle (1982), wherein it is found that certain amino acids may be substituted for other amino acids having a similar hydropathic index or core and still retain a similar biological activity. As displayed in Table B below, amino acids are assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. It is believed that the relative hydropathic character of the amino acid determines the secondary structure of the resultant protein, which in turn defines the interaction of the protein with substrate molecules. Preferred substitutions which result in an antigenically equivalent peptide or protein will generally involve amino acids having index scores within 2 units of one another, and more preferably within 41 unit, and even more preferably, within 0. 5 units.

Table B Amino Acid Hydropathic Index Isoleucine 4. 5 Valine 4. 2 Leucine 3. 8 Phenylalanine 2. 8 Cysteine/cystine 2. 5 Methionine 1. 9 Alanine 1. 8 Glycine-0. 4 Threonine-0. 7 Tryptophan-0. 9 Serine-0. 8 Tyrosine-1. 3 Proline-1. 6 Histidine-3. 2 Glutamic Acid-3. 5 Glutamine-3. 5

Table B (continued) Aspartic Acid-3. 5 Asparagine-3. 5 Lysine-3. 9 Arginine-4. 5 Thus, for example, isoleucine, which has a hydropathic index of +4. 5, will preferably be exchanged with an amino acid such as valine (+ 4. 2) or leucine (+ 3. 8).

Alternatively, at the other end of the scale, lysine (-3. 9) will preferably be substituted for arginine (-4. 5), and so on.

Substitution of like amino acids may also be made on the basis of hydrophilicity, particularly where the biological functional equivalent protein or peptide thereby created is intended for use in immunological embodiments. U. S.

Patent 4, 554, 101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i. e. with an important biological property of the protein.

As detailed in U. S. Patent 4, 554, 101, each amino acid has also been assigned a hydrophilicity value. These values are detailed below in Table C.

Table C Amino Acid Hydrophilic Index arginine +3. 0 lysine +3. 0 aspartate +3. 01 glutamate +3. 0 1 serine +0. 3 asparagine +0. 2

Table C (continued) glutamine +0. 2 glycine 0 threonine-0. 4 alanine-0. 5 histidine-0. 5 proline-0. 5 1 cysteine-1. 0 methionine-1. 3 valine-1. 5 leucine-1. 8 isoleucine-1. 8 tyrosine-2. 3 phenylalanine-2. 5 It is understood that one amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein. In such changes, the substitution of amino acids whose hydrophilicity values are within 2 is preferred, those which are within il are particularly preferred, and those within 40. 5 are even more particularly preferred.

Accordingly, these amino acid substitutions are generally based on the relative similarity of R-group substituents, for example, in terms of size, electrophilic character, charge, and the like. In general, preferred substitutions which take various of the foregoing characteristics into consideration will be known to those of skill in the art and include, for example, the following combinations : arginine and lysine ; glutamate and aspartate ; serine and threonine ; glutamine and asparagine ; and valine, leucine and isoleucine.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include : arginine and lysine ; glutamate and aspartate ; serine and threonine ; glutamine and asparagine ; and valine, leucine and isoleucine (See Table D, below). The present invention thus contemplates functional or biological equivalents of an UCH-L3 or variant UCH-L3 polypeptide as set forth above.

Table D Original Residue Exemplary Substitutions Ala Gly ; Ser Arg Lys Asn Gln ; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Ala His Asn ; Gln Ile Leu ; Val Leu Ile ; Val Lys Arg Met Met ; Leu ; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp ; Phe

Original Residue Exemplary Substitutions Val Ile ; Leu Biological or functional equivalents of a polypeptide can also be prepared using site-specific mutagenesis. Site-specific mutagenesis is a technique useful in the preparation of second generation polypeptides, or biologically functional equivalent polypeptides or peptides, derived from the sequences thereof, through specific mutagenesis of the underlying DNA. As noted above, such changes can be desirable where amino acid substitutions are desirable. The technique further provides a ready ability to prepare and test sequence variants, for example, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.

Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by Adelman, et al. (1983). As will be appreciated, the technique typically employs a phage vector which can exist in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the M13 phage (Messing, et al., 1981). These phage are commercially available and their use is generally known to those of skill in the art.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector which includes within its sequence a DNA sequence which encodes all or a portion of the UCH-L3 or variant UCH-L3 enzyme polypeptide sequence selected. An oligonucleotide primer bearing the desired

mutated sequence is prepared, generally synthetically, for example, by the method of Crea et al. (1978). This primer is then annealed to the singled-stranded vector, and extended by the use of enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells such as E. coli cells and clones are selected which include recombinant vectors bearing the mutation. Commercially available kits come with all the reagents necessary, except the oligonucleotide primers.

In addition, peptides derived from these polypeptides, including peptides of at least about 6 consecutive amino acids from these sequences, are contemplated. Alternatively, such peptides may comprise about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 consecutive residues. For example, a peptide that comprises 6 consecutive amino acid residues may comprise residues 1 to 6, 2 to 7, 3 to 8 and so on of the UCH-L3 protein.

Such peptides may be represented by the formula x to (x + n) = 5'to 3'the positions of the first and last consecutive residues where x is equal to any number from 1 to the full length of the UCH-L3 protein and n is equal to the length of the peptide minus 1. Where the peptide is 10 residues long (n = 10-1), the formula represents every 10-mer possible for each antigen. For example, where x is equal to 1 the peptide would comprise residues 1 to (1 + [10-1]), or I to 10. Where x is equal to 2, the peptide would comprise residues 2 to (2 + [10-2]), or 2 to 11, and so on.

Syntheses of peptides are readily achieved using conventional synthetic techniques such as the solid phase method (e. g., through the use of a commercially

available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptides synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may be readily stored in aqueous solutions for fairly long periods of time if desired, e. g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity. However, where extended aqueous storage is contemplated it will generally be desirable to include agents including buffers such as Tris or phosphate buffers to maintain a pH of 7. 0 to 7. 5. Moreover, it may be desirable to include agents which will inhibit microbial growth, such as sodium azide or Merthiolate. For extended storage in an aqueous state it will be desirable to store the solutions at 4°C, or more preferably, frozen. Of course, where the peptide (s) are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e. g., in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled, deionized) or buffer prior to use.

Of particular interest are peptides that represent antigenic epitopes that lie within the UCH-L3 polypeptides of the present invention. An"epitope"is a region of a molecule that stimulates a response from a T-cell or B-cell, and hence, elicits an immune response from these cells. An epitopic core sequence, as used herein, is a relatively short stretch of amino acids that is structurally"complementary"to, and therefore will bind to, binding sites on antibodies or T-cell receptors. It will be understood that, in the context of the present disclosure, the term"complementary" refers to amino acids or peptides that exhibit an attractive force towards each other.

Thus, certain epitopic core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the corresponding UCH-L3 antigen to the corresponding UCH-L3-directed antisera.

The identification of epitopic core sequences is known to those of skill in the art. For example U. S. Patent 4, 554, 101 teaches identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity, and by Chou- Fasman analyses. Numerous computer programs are available for use in predicting antigenic portions of proteins, examples of which include those programs based upon Jameson-Wolf analyses (Jameson and Wolf, 1988 ; Wolf et al., 1988), the program PepPlott) (Brutlag et al., 1990 ; Weinberger et al., 1985), and other new programs for protein tertiary structure prediction (Fetrow and Bryant, 1993) that can be used in conjunction with computerized peptide sequence analysis programs.

In general, the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences. The smallest useful core sequence expected by the present disclosure would be on the order of about 6 amino acids in length. Thus, this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention.

However, the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.

Small Molecule Inhibitors of UCH-L3 Variant Proteins The present invention provides methods for screening and identifying small molecule inhibitors of UCH-L3 proteins and identifies such inhibitors. The rationale behind the design of the small molecule UCH-L3 protein inhibitors is that the structural differences between UCH-L3 proteins, caused by the deviations in the interatomic distances of the amino acid residues in the active site of the protein, will be exploited to design chemical ligands that bind to the active site of the different variant proteins to yield complexes with sufficient thermodynamic stability to effectively inhibit the functional activity of the protein. The inhibited UCH-L3 protein is thus unable to protect the tumor cell against the toxic action of the anticancer agent used to treat it. To obtain appropriate ligands that bind to the active sites of different UCH-L3 variant proteins, the inventors utilize the technique of

forcefield docking of chemical fragments from both commercially available chemical fragment libraries, as well as in-house generated libraries, into the active electrophile- binding (H-) site in the derived crystal structure of each variant protein. The docked fragments will be energy-minimized and the binding energies computed and used to select candidate ligands.

Generation of UCH-L3 Inhibitors Generation of inhibitors is accomplished by a rational drug development strategy involving force field docking and energy-minimization of chemical fragments and compounds into the active site of the variant UCH-L3 proteins. The compounds and chemical fragments can be drawn from chemical fragment libraries, such as that available in the Leapfrog database. Additional chemical libraries will be generated as necessary. The active site and other structural components of the variant UCH-L3 proteins will be derived from the published crystal structure of the UCH-L3 encoded protein.

One potential substitution that confers a functional change to the UCH-L3 protein is to replace cysteine 95 with a serine that, in context with other such changes, results in a protein that is more chemically stable and resistant to oxidation and heat.

Other proposed changes in this context include substituting aspartic acid 184 for asparagine. Moreover, it is recognized that leucine may be substituted for methionine, or a serine or alanine may be substituted for cystine to result in increased stability.

Increased protein stability also results from the addition of disulfide bonds and the creation of more hydrophobic interactions within the protein structure.

Based on the resultant DDH values obtained after energy minimization of chemical fragments/compounds, candidate inhibitors are selected and/or newly constructed from chemical fragments for synthesis and further analyses for their inhibitory or other action on the variant UCH-L3 proteins. Selection criteria for

inhibitors for synthesis and further analysis includes lipophilicity, chemical stability and availability or ease of synthesis.

Candidate inhibitors of the present invention may include such molecules as substituted, heterocyclic aromatic compounds, sugar-linked aromatic compounds and other aromatic compounds.

The substituted groups may vary between the different compounds and result in significant changes in binding energies of the compounds in the active site pocket of the UCH-L3 protein. For example, Rl substitutions of either NH2 or OH, cause changes in binding energies of almost 10 kcals/mol. Other important substitutions are the alkyl or aminoalkyl substitutions of R3, and the alkyl, phenyl or 2-pyridyl substitutions of R4, some of which result in changes in binding energies of greater than 10 kcals/mol.

However it is conceivable that any of the R groups of the substituted isoxazoles may be a phenyl group, a benzyl group, an aryl group, an alkyl group, an aryl group linked to another aryl group through an ester linkage, an aryl group linked to an alkyl group with an ester linkage, an aryl group linked to another aryl group through an ether linkage and aryl group linked to an alkyl group with a thiolester linkage, an alkyl group linked to another alkyl group through an ester linkage, an alkyl group linked to another alkyl group through an ether linkage, an alkyl to alkyl linked through an amino group, an aryl to alkyl linked through an amino group. an alkyl group through a disulphide group, an aryl linked to an alkyl group through a disulphide group, an aryl linked to another aryl group through a disulphide group, an alkyl linked to another alkyl group through a thioester linkage, an aryl linked to an alkyl group through a polyester linkage, an aryl group linked to another aryl through a polyester linkage, an alkyl group linked to another alkyl group through a polyamine linkage, an aryl linked to an alkyl group through a polyamine linkage, an aryl group linked to another aryl through a polyamine linkage, an alkyl group linked to another

alkyl group through a polythioester linkage, an aryl linked to an alkyl group through a polythioester linkage, an aryl group linked to another aryl through a polythioester linkage.

An individual skilled in the art of organic synthesis in light of the present disclosure is able to prepare or identify a large variety of substituted isoxazoles which would be expected to have UCH-L3 inhibitory effects in the light of the present disclosure.

Screeningfor Modulators of UCH-L3.

Within certain embodiments of the invention, methods are provided for screening for modulators of UCH-L3 protein activity. Such methods may use labeled UCH-L3 proteins or analogs, anti-UCH-L3 proteins or anti-UCH-L3 antibodies and the like as reagents to screen small molecule and peptide libraries to identify modulators of UCH-L3 protein activity. Within one example, a modulator screening assay is performed in which cells expressing UCH-L3 proteins are exposed to a test substance under suitable conditions and for a time sufficient to permit the agent to effect activity of UCH-L3 proteins.

Assay for Ubiquitin Carboxy Terminal Hydrolase Activity To perform the assay, purified UCH-L3 or variant UCH-L3 peptide is diluted into 10 mM dithiothreitol (DTT) and allowed to preincubate on ice for 1 h. The standard assay contains 12 uM UbOEt (ubiquitin carboxy-terminal ethyl ester), 100 mM potassium phosphate, pH 7. 2 (37°C), 10 mM dithiothreitol, 0. 2 mM EDTA, and enzyme diluted to a final concentration of 0. 4 mIU/ml. The reaction is incubated at 37°C and aliquots containing 1-2 g total ester plus hydrolysis product are withdrawn at ten minute intervals and immediately injected onto an HPLC column (C-8, 5 micron, 4 mm x 250 mm ; Altech Associates, Deerfield, IL), flow rate of 1 ml/min in a solvent comprising 25 mM sodium Perchlorate and 0. 07% (v/v)

perchloric acid in 49% HPLC grade acetonitrile. The absorbance at 205 nm is monitored and the resulting peaks are quantitated by manual integration of the areas.

Measurement of Deconjugating Activity 125 I-ubiquitin was synthesized by the chloramine-T method (Ciechanover et al. 1978, Biochem. Biophys. Res. Comm. 81, 1100-1104). 1-ubiquitin was conjugated to the proteins of reticulocyte fractions by incubating the following in a final volume of 0. 8 ml : 2 mg/ml of proteins, 3 pg/ml 125 I-ubiquitin (1. 2 x 106 cpm/lFlg), 50 mM Tris HCI, pH 7. 6, 1 mM magnesium chloride, 0. 4 mM ATP, 0. 4 mM DTT, 2 mM phosphocreatine, 3 units creatine phosphokinase, and 0. 1 mM hemin.

After incubating 2 h at 37°C, iodoacetamide was added to a final concentration of 10 mM, and was allowed to react for 30 min. at 37°C. Dithiothreitol was then added to a concentration of 50 mM to quench the alkylating agent, and the mixture was chromatographed on a Sephadex G-50 column (1. 5 cm x 60 cm) equilibrated with 50 mM ammonium acetate. The fractions containing 125 I-ubiquitin were located by gamma counting, and the counts in the exclusion volume are pooled. The percentage of ubiquitin incorporated into high molecular weight complexes is about 10%, and this fraction is utilized in deconjugation assays.

To measure deconjugation, the 125 I-ubiquitin conjugates are incubated with 0. OIU of the UCH fraction in 50 mM Tris HCl pH 8. 0, 0. 1 mM EDTA, and 10 mM DTT. After 30 min. or 2 h, the reaction is terminated by the addition of two parts reaction mixture to one part 9% SDS, 15% glycerol, 0. 2 M Tris Hcl, pH 6. 8, and 3 mM EDTA. The samples are then subjected to SDS-PAGE according to standard techniques. The resulting gels dried and sliced into strips. The molecular weight distribution of 125 I-ubiquitin is determined by gamma counting of the gel slices.

Rates of deconjugation is calculated by the fraction of counts appearing in the sub- 10 kD region relative to the entire lane.

Generally the test substance is added in the form of a purified agent, however it is also contemplated that test substances useful within the invention may include substances present throughout the handling of test sample components, for example host cell factors that are present in a cell lysate used for generating the test sample. Such endogenous factors may be segregated between the test and control samples for example by using different cell types for preparing lysates, where the cell type used for preparing the test sample expresses a putative test substance that is not expressed by the cell type used in preparing the control sample.

The active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive. However, prior to testing of such compounds in humans or animal models, it may be necessary to test a variety of candidates to determine which have potential.

Accordingly, in screening assays to identify agents which alter the activity of UCH-L3 proteins in for example cancer cells, it is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds.

In these embodiments, the present invention is directed to a method for determining the ability of a candidate substance to decrease the UCH-L3 activity of cancer cells, the method including generally the steps of : (a) obtaining a cell with UCH-L3 activity ; (b) admixing a candidate substance with the cell ; and

(c) determining the ability of the candidate substance to inhibit the UCH- L3 activity of the cell.

To identify a candidate substance as being capable of decreasing UCH-L3 activity, one would measure or determine the basal UCH-L3 status of for example a cancer cell prior to any additions or manipulation. One would then add the candidate substance to the cell and re-determine the UCH-L3 activity in the presence of the candidate substance. A candidate substance which decreases the UCH-L3 activity relative to the composition in its absence is indicative of a candidate substance being an inhibitor of UCH-L3.

The candidate screening assay is quite simple to set up and perform, and is related in many ways to the assay discussed above for determining UCH-L3 content.

"Effective amounts", in certain circumstances, are those amounts effective at reproducibly decrease UCH-L3 activity in an assay in comparison to their normal levels. Compounds that achieve significant appropriate changes in activity will be used. If desired, a battery of compounds may be screened in vitro to identify other agents for use in the present invention.

A significant decreases in UCH-L3 activity, are represented by a decrease in UCH-L3 protein activity levels of at least about 30%-40%, and most preferably, by decreases of at least about 50%, with higher values of course being possible. Assays that measure UCH-L3 activity in cells are well known in the art and may be conducted in vitro or in vivo, and have been described elsewhere in the specification.

Quantitative in vitro testing of the UCH-L3 inhibitors is not a requirement of the invention as it is generally envisioned that the agents will often be selected on the basis of their known properties or by structural and/or functional comparison to those agents already demonstrated to be effective. Therefore, the effective amounts will

often be those amounts proposed to be safe for administration to animals in another context.

EXAMPLE 1 : SMALL MOLECULE INHIBITORS OF UCH-L3 AND UCH-L3 VARIANTS 1. Materials and Methods Generation of UCH-L3 inhibitors. Generation of inhibitors is accomplished by a rational drug development strategy involving force field docking and energy- minimization of chemical fragments and compounds into the active site of the variant UCH-L3 proteins. The compounds and chemical fragments can be drawn from chemical fragment libraries, such as that available in the Leapfrog database. Additional chemical libraries will be generated as necessary. The active site and other structural components of the variant UCH-L3 proteins will be derived from the published crystal structure of the UCH-L3 encoded protein. Selection criteria for inhibitors for synthesis and further analysis includes lipophilicity, chemical stability and availability or ease of synthesis.

Synthesis of UCH-L3 Inhibitors. If the identified and/or newly constructed potential inhibitors are not commercially available, then they will be synthesized using standard organic synthetic methodology, including heterocyclic ring construction and functionalization, and electrophilic and nucleophilic substitution reactions. Reaction mixtures will be separated by thin layer, flash silica gel column and high performance liquid chromatography (TLC, CC and HPLC). The compounds will be purified using standard techniques modified as necessary. Characterization of synthetic products will be done by melting point determination, Fourier transform infrared (FT-1R), ultraviolet (UV) and high resolution nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Compounds for biological testing will be purified by preparative HPLC. The purity of compounds will be determined by elemental analysis and HPLC.

Source of variant UCH-L3 proteins. To examine the ability of the inhibitors selected from the rational design described above to inhibit the variant UCH-L3 proteins, the present invention will utilize recombinant UCH-L3 proteins expressed in E. coli transfected with expression vectors containing the corresponding cDNAs.

These vectors have been described elsewhere in this application. The UCH-L3 proteins will be purified by GSH-affinity chromatography on S-hexyl glutathione linked to epoxy-activated sepharose 6B. and then used for enzyme kinetic analysis.

It is also recognized that one may employ a ubiquitin affinity column to purify the UCH-L3 proteins and their variants. In this method, UCH-L3 and or variant UCH-L3 is contacted with activated CH-Sepharose 4B to which ubiquitin is bound.

The enzyme forms a thiol ester linkage to the bound ubiquitin in the presence of ATP and is eluted with AMP plus inorganic pyrophosphate. The amount of functional enzyme is determined from the counts of (3H) ATP made acid insoluble by formation of 1 enzyme equivalent of (3H) AMP-ubiquitin. Treatrnent of the activating enzyme with iodoacetamide renders it unable to form Es-ubiquitin but has no effect on formation of E-AMP-ubiquitin (Ross and Warms, Biochemistry, 22 : 4234-4237 (1983).

Analysis of inhibitors for UCH-L3 inhibitory activity. These studies will be performed using standard enzyme kinetic methodologies. The purified variant UCH- L3 proteins will be mixed with increasing inhibitor concentrations and at different time points, residual deubiquitinating activity will be determined as set forth above.

Synthesis of Isoxazoles. Using the techniques described above, potential UCH-L3 inhibitors such as isoxazoles have been identified. In the synthetic strategy for obtaining isoxazole deubiquitinating inhibitors, the ring system can be acnieved by the usual approach of cyclization between hyroxylamine and three-carbon atom component such as 1, 3-diketone or an ab-unsaturated ketone or by a 1, 3-dipolar

cycloaddition reaction involving nitride oxides with alkenes or an alkyne (Glichrist, 1992).

The first class of compounds are substituted isoxazoles, with the general structure shown in structures 1-3. The substituted groups in the different compounds are represented by Rl, R2, R3, and R4. The substituted groups vary between the different compounds and result in significant changes in binding energies of the compounds in the active site pocket of the UCH-L3 protein. For example, Rl substitutions of either NH2 or OH, cause changes in binding energies of almost 10 kcals/mol. Other important substitutions are the alkyl or aminoalkyl substitutions of R3, and the alkyl, phenyl or 2-pyridyl substitutions of R4, some of which result in changes in binding energies of greater than 10 kcals/mol.

Another group of potential variant UCH-L3 protein inhibitors identified by the strategy described in this invention are the heterocyclic aromatic compounds. The binding energies range from-34 to-94 kcal/mol, depending upon the type of compound or substitution.

Antibodies Antibodies to UCH-L3 or UCH-L3 variant peptides or polypeptides may be readily prepared through use of well-known techniques, such as those exemplified in U. S. Patent 4, 196, 265. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e. g., purified or partially purified protein, synthetic protein or fragments thereof, as discussed in the section on polypeptides. Animals to be immunized are mammals such as cats, dogs and horses, although there is no limitation other than that the subject be capable of mounting an immune response of some kind. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is possible. The use of rats may provide certain advantages, but mice are preferred, with the BALB/c

mouse being most preferred as the most routinely used animal and one that generally gives a higher percentage of stable fusions.

For generation of monoclonal antibodies (MAbs), following immunization, somatic cells with the potential for producing antibodies, specifically p lymphocytes cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of the animal with the highest antibody titer removed. Spleen lymphocytes are obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 5 x 107 to 2 x lOB lymphocytes.

The antibody-producing B cells from the immunized animal are then fused with cells of an immortal myeloma cell line, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells, called"hybridomas." Any one of a number of myeloma cells may be used and these are known to those of skill in the art. For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8. 653, NSl/l. Ag 41, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1. 7 and S194/5XXO Bul ; for rats, one may use R210. RCY3, Y3-Ag 1. 2. 3, IR983F and 4B210 ; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell line is the NS-1 myeloma cell line (also termed P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cell line.

Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2 : 1 proportion, though the proportion may vary from about 20 : 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai virus have been described by Kohler and Milstein (1975 ; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate.

Fusion procedures usually produce viable hybrids at low frequencies, from about 1 x 10-6 to 1 x 10-8. This does not pose a problem, however, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culture in a selective medium. The selective medium generally is one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.

Exemplary and preferred agents are aminopterin, methotrexate and azaserine.

Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells

are defective in key enzymes of the salvage pathway, e. g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas are then serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways. A sample of the hybridoma can be injected, usually in the peritoneal cavity, into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration. The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations. MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.

Monoclonal antibodies of the present invention also include anti-idiotypic antibodies produced by methods well-known in the art. Monoclonal antibodies

according to the present invention also may be monoclonal heteroconjugates, i. e., hybrids of two or more antibody molecules. In another embodiment, monoclonal antibodies according to the invention are chimeric monoclonal antibodies. In one approach, the chimeric monoclonal antibody is engineered by cloning recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse antibody producing cell and the constant-region exons from a human antibody gene.

The antibody encoded by such a recombinant gene is a mouse-human chimera. Its antibody specificity is determined by the variable region derived from mouse sequences. Its isotype, which is determined by the constant region, is derived from human DNA.

In another embodiment, monoclonal antibodies according to the present invention is a"humanized"monoclonal antibody, produced by techniques well-known in the art. That is, mouse complementary determining regions ("CDRs") are transferred from heavy and light V-chains of the mouse Ig into a human V-domain, followed by the replacement of some human residues in the framework regions of their murine counterparts."Humanized"monoclonal antibodies in accordance with this invention are especially suitable for use in in vivo diagnostic and therapeutic methods for treating Moroxella infections.

As stated above, the monoclonal antibodies and fragments thereof according to this invention can be multiplied according to in vitro and in vivo methods well-known in the art. Multiplication in vitro is carried out in suitable culture media such as Dulbecco's modified Eagle medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements, e. g., feeder cells, such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like. In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for large scale hybridoma cultivation under tissue culture conditions are known in the art and include homogenous suspension culture, e. g, in

an airlift reactor or in a continuous stirrer reactor or immobilized or entrapped cell culture.

Large amounts of the monoclonal antibody of the present invention also may be obtained by multiplying hybridoma cells in vivo. Cell clones are injected into mammals which are histocompatible with the parent cells, e. g., syngeneic mice, to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially oils such as Pristane (tetramethylpentadecane) prior to injection.

In accordance with the present invention, fragments of the monoclonal antibody of the invention can be obtained from monoclonal antibodies produced as described above, by methods which include digestion with enzymes such as pepsin or papain and/or cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer, or they may be produced manually using techniques well known in the art.

The monoclonal conjugates of the present invention are prepared by methods known in the art, e. g., by reacting a monoclonal antibody prepared as described above with, for instance, an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Conjugates with fluorescein markers are prepared in the presence of these coupling agents, or by reaction with an isothiocyanate. Conjugates with metal chelates are similarly produced. Other moieties to which antibodies may be conjugated include radionuclides such as 3H, l25I, l3lI 32p, 35S, 14C, SlCr, 36Cl, 57Co, 5eco, Se, Eu, and 99mTc, are other useful labels which can be conjugated to antibodies. Radioactively labeled monoclonal antibodies of the present invention are produced according to well-known methods in the art. For instance, monoclonal antibodies can be iodinated by contact with sodium or potassium iodide and a chemical oxidizing agent such as sodium hypochlorite, or an enzymatic oxidizing

agent, such as lactoperoxidase. Monoclonal antibodies according to the invention may be labeled with technetium-99m by ligand exchange process, for example, by reducing pertechnate with stannous solution, chelating the reduced technetium onto a Sephadex column and applying the antibody to this column or by direct labeling techniques, e. g., by incubating pertechnate, a reducing agent such as SNC12, a buffer solution such as sodium-potassium phthalate solution, and the antibody.

The present invention contemplates that the exclusion of large ubiquitin fusions from the UCH-L3 active site results from the 20 residue loop between Thr- 147 and Val-166 that is disordered in the instant crystals. This loop is topologically distinct from the papain-like enzymes. The ends of the loop are anchored 20 A apart on opposite sides of the active site Cys-95 and three different classes of conformations can be envisioned for the loop with respect to the proposed UCH-substrate interaction geometry (FIG. 9).

The loop may be sandwiched between the body of UCH-L3 and the ubiquitin moiety of a substrate (red conformation in FIG. 9). This arrangement seems unlikely, however, in light of the probable ubiquitin binding surface on UCH-L3 (see above).

Furthermore, the loop sequence is not well conserved, and thus seems poorly suited to mediate interactions with ubiquitin, for which all UCH enzymes that have been characterized exhibit high specificity.

A second possible conformation places the loop over the active site, with residues C-terminal to the scissile bond passing through the loop (blue in FIG. 9).

When modeled in a maximally open conformation the loop has an internal diameter of approximately 15 A, which is suitable for passage of an unfolded extended polypeptide chain, although it is expected to limit passage of even a small folded structure such as an a-helix. A problem with this model is that the D. melanogaster UCH is able to cleave ubiquitin from conjugates with the large substrate IlcBa (Roff

et al., 1996), and that the S. cerevisiae UCH cleaves conjugates from cytochrome c (Cohen).

Alternatively, the disordered loop may fold completely away from the proposed ubiquitin-binding surface (magenta in FIG. 9). This conformation would be analogous to the occluding loop of cathepsin B, which is also located along the S' sites and defines the exopeptidase specificity of cathepsin B by making specific interactions with the substrate carboxyl terminus two residues beyond the scissile bond (Turk et al., 1995). An important topological distinction is that, unlike the disordered loop of UCH-L3, the cathepsin B occluding loop does not straddle the active cleft site (in FIG. SA the occluding loop partially obscures the active site Gin, Cys, and His of cathepsin B).

The present invention contemplates changing the topology of the UCH-L3 protein to make it more papain-like in structure, such that the resulting protein is capable of cleaving peptides as well as larger proteins from ubiquitin. In constructing such a molecule, the disordered loop that straddles the active site is reduced or eliminated, thus opening the active site.

It is possible that upon binding of ubiquitin adducts, the disordered loop will remain mobile, fluctuating between the extreme vertical line and horizontal line conformations of FIG. 9. Thus, the loop will impede active site access for a wide range of larger substrates, which may eventually attain a productive complex by using either the horizontal line or vertical line conformations. It is also possible that the disordered loop plays a more active role in the selection of substrates in vivo, perhaps even becoming ordered and contributing directly to binding of some physiological substrates. This model suggests the intriguing possibility that the disordered loops of the different UCH enzymes, which are of similar length but relatively dissimilar sequence identities, function as modular units to confer different substrate specificity on the various UCH isozymes.

Regulating protein degradation by regulating protein deubiquitination can be stimulating or inhibiting degradation. Where protein degradation is to be stimulated a protein whose degradation is ubiquitin-dependent is exposed to a UCH-L3 or mutant UCH-L3 enzyme of the present invention.

Where protein degradation is to be inhibited, a protein whose degradation is ubiquitin-dependent is exposed to a mutant deubiquitinating enzyme of the present invention, which mutant does not catalyze the deubiquitination of proteins.

Exposing can be accomplished in vitro or in vivo. In vitro deubiquitinating processes have application in the industrial bulk production of proteins such as enzymes. A deubiquitinating enzyme of the present invention can be used in such processes to remove ubiquitin from the produced protein or to direct the removal of selected terminal amino acid residues. The use of deubiquitinating enzymes for generating desired amino-terminal residues of proteins is described in United States Patent No. 5, 093, 242, the disclosure of which is incorporated herein by reference.

Where exposing is accomplished in vivo, cells lacking an endogenous deubiquitinating system or cells having a mutation or deficiency in a deubiquitinating enzyme are transfected with a polynucleotide comprising a DNA sequence that encodes a deubiquitinating enzyme. Alternatively, a cell can be transfected with an expression vector comprising a DNA sequence that encodes a mutant UCH-L3 such that the natural protein degradation pathway for a protein is inhibited.

Processes for destabilizing proteins in vivo, producing proteins using ubiquitin fusion and the in vitro cleavage of ubiquitin fusion proteins are well known in the art.

Descriptions of such processes can be found in United States Patent Nos. 5, 122, 463, 5, 132, 213 and 5, 196, 321, the disclosures of which are incorporated herein by reference. In addition, the nucleotide and amino acid residue sequences of ubiquitin-

specific proteases can be found in United States Patent No. 5, 212, 058, the disclosure of which is incorporated herein by reference.

EXAMPLE 2 : SPECIFICITY AND IN VIVO ROLES OF UCH ISOZYMES To further define the specificity and the in vivo roles of UCH isozymes, the present inventors tested natural and semi-synthetic ubiquitin derivatives as substrates, with specific emphasis on their potential role in ubiquitin proprotein and polyubiquitin processing. The results suggest that human UCH isozymes L1 and L3 are apparently involved in processing of proubiquitin gene products and small molecular weight ubiquitin adducts, but not larger derivatives of ubiquitin.

Procedures Materials Ubiquitin C-terminal hydrolases were prepared as described previously (Larsen etaL, 1996). All chemicals were reagent grade or better. Restriction endonucleases and DNA modification enzymes were from New England Biolabs, Beverly, MA. Recombinant human ubiquitin was expressed in E. coli and purified as described below.

Subcloning ofproprotein genes The human UbCEP52 and UbCEP80 and the S. cerevisiae ubi4 proubiquitin genes were excised from pSP72 cloning vector (Monia et aL, 1989) by digestion with EcoRV and KpnI. The cassette was ligated to a 5'NdeI site (Klenow polymerase blunted) and the 3'KpnI site of the prokaryotic expression vector pRSET B (Invitrogen) with T4 DNA ligase. After transformation of the ligation mixture to Top 10 F'competent E. coli (Invitrogen), clones were grown for DNA miniprep and assayed by restriction digestion with ScaI and XhoI (UbCEP52) or ScaI and BamHI (UbCEP80). Correct recombinant plasmids were amplified and stored at-20°C in TE

buffer (Sambrook etal., 1989). The yeast proubiquitin gene was similarly inserted into the Klenow-blunted pRSET using EcoRV and HindIII, and colonies were screened with by XhoI restriction digests of the isolated plasmids. These ubiquitin proprotein expression plasmids were named pRSUb52, pRSUb80, or pRSyUbS, respectively.

Purification of ubiquitin proproteins The E. coli host strain BL21 (DE3) (Invitrogen) was transformed with the appropriate expression vectors described above. For Ub-CEP proteins, the strain BL21 (DE3) pLysE was used. Individual colonies were inoculated into 200 ml LB media (Sambrook et al., 1989) supplemented with ampicillin (50 llg/ml) and grown overnight at 37°C. This culture was used to inoculate 2 or 12 liters of LB media.

When the optical density (600 nm) of the cultures reached 0. 45 (UbCEP) or 0. 6 (yUb5), IPTG was added to 0. 3 mM, and the cultures were grown for an additional 3 h. The cells were pelleted at 4000 RPM in an RC-3 rotor. Lysozyme was added to 0. 1 mg/ml, and the bacteria were incubated for thirty minutes at 37°C, sonicated, and recentrifuged as above. UbCEP52 was purified from the supernatant as described previously (Monia et al., 1989), with additional purification over a 300 ml sephadex G-75SF gel filtration column and MonoS FPLC (Pharmacia) Recombinant yeast proubiquitin was expressed in E. coli and purified by a modification of Jonnalagadda et al. (1987). The bacteria were harvested by centrifugation, suspended in 50 mM Tris-Cl, pH 7. 8, 1 mM EDTA, and sonicated (Heat Systems). After centrifugation for thirty minutes at 15, 000 x g, the supernatant was raised to 65°C for five minutes, and centrifuged again as above. The resulting heat stable supernatant was made 85% saturated in ammonium sulfate, stirred gently overnight at 4°C, and was centrifuged for thirty minutes at 10, 000 x g in a GSA rotor.

The pellet was resolubilized in a minimal volume of water, and after lowering its pH to 4. 6 with 1 M acetic acid, was applied to an FPLC Mono S 5/5 column (Pharmacia) in 50 mM NaOAc pH 4. 5. Ubiquitin oligomers were eluted in a linear gradient of 0

to 550 mM NaCl. Oligomers which cross reacted with anti-ubiquitin polyclonal antibodies (Accurate Scientific) eluted at 150, 200, 290, 350, and 400 mM NaCl (n= 1 to 5 ubiquitins respectively). The pooled fractions were dialyzed against 10 mM Tris- Cl, pH 7. 6, concentrated by ultrafiltration. The preparation was homogeneous as judged by coomassie-stained SDS-PAGE.

Purification of truncated ubiquitin gene products To study P'specificity, the truncated ubiquitin gene products, Ub-CEP52 Ub-CEP80 1-10, and Ub-Ub-l° were prepared. Vectors encoding ubiquitin fused to the first ten residues of CEP52 (Ub-IIEPSLRQLA) (SEQ ID NO : 1), CEP80 (Ub-GKKRKKKVYT) (SEQ ID NO : 2), or Ub (Ub-MQIFVKTLTG) (SEQ ID NO : 3). Bacteria harboring the expression plasmids were grown to an A600 of 0. 6, and induced for protein production with 0. 5 mM IPTG. Supernatants were made as above, but with 10 mM DTT in the buffer. The supernatants containing Ub-CEP521-10 or the Ub-CEP80l-l° were heat treated at 86°C for five minutes, cooled to 4°C, and centrifuged at 3, 500 x g for 15 min. In most cases, the supernatant was chromatographed on a 1 liter column of G-100 superfine (Pharmacia). The supernatant containing Ub-Ubluo was pretreated with 2. 5% perchloric acid and centrifuged. The acid-soluble supernatant was subjected to gel filtration as above. In all cases, the fusion proteins obtained were homogeneous as judged by Coomassie- stained SDS-PAGE.

Preparation of Ub-amino acid extension proteins A vector library encoding a variety of single amino acids C-terminal to ubiquitin was constructed using the polymerase chain reaction. To create this amino acid library at position 77, the coding region of the pRSUb80 vector (see above) was amplified with a degenerate 3'primer which contained all possible codons followed by a stop codon and a HindIII site. The primer sequences were : 5'-ATCCATATGCAGATCTTCG-3' (SEQ ID NO : 4), and

5'-CAAGCTTCTANNNACCACCACGAAGTC-3' (SEQ ID NO : 5). The PCRTM products from this reaction were subcloned en masse into pCRII (Invitrogen, San Diego, CA), and 40 minipreps were prepared. Inserts were present in 25 of the 40 minipreps and these inserts were sequenced (Sanger et al., 1977). Clones were identified which encoded D, H, K, P, S, or T at the C-terminus. These were subcloned into pRSET using their NdeI and HindIII sites. Proteins were expressed and purified by heat denaturation and gel filtration, as described above. One additional clone was recovered due to a deletion in the PCRTM product. This frameshift resulted in a vector encoding Na-ubiquitinyl-PRSLDSC, which was also expressed and purified.

Co-translational processing The kanamycin resistance gene was incorporated into plasmids encoding UCH-L1 or UCH-L3 by insertion of a DNA cassette from pUC4K (Pharmacia). pRSUCH plasmids were digested with EcoRI and calf intestinal phosphatase. The kanr gene cassette was excised from pUC4K with EcoRI. After gel purification of the insert and vector fragments, they were ligated and plated onto LB-kanamycin agar plates. The correct transformants were identified by the presence of a unique ScaI site in the kanr cassette, and the ampr gene was subsequently disabled by excision of an AvaII fragment in its center, followed by religation.

To co-express enzymes and putative substrates in the same cell, BL21 (DE3) cells harboring either the pRSyUb5, the pRSUb52, or the pRSUb80 plasmid (ampr) were transformed with a pRSUCH plasmid (kanr) and plated on LB agar containing both kanamycin and ampicillin to select for co-transformants. Induction with IPTG resulted in co-expression of the selected UCH isozyme along with a putative substrate. Processing was assessed by adding SDS-PAGE sample buffer directly to cell pellets and analyzed by Western blotting using antibodies specific for each substrate.

Other substrates A plasmid encoding a Ub-R-p-galactosidase fusion protein was obtained from Dr. Alex Varshavsky (pKKUbRpGal). Synthesis of Ub-R-p-gal was induced as described previously, and the fusion protein was purified as described for UCH, except that the anion exchange resin was eluted with 50 mM Tris-HCl, pH 7. 5 and containing 150 mM NaCl. This resulted in significantly purified protein preparation (>80% homogeneous) which was used in gel and HPLC assays of fusion protein processing.

K48-linked diubiquitin (Ne-Ubiquitinyl-K 8Ub) was synthesized in vitro by incubation of human recombinant or bovine ubiquitin (Sigma, St. Louis, MO) with the activating and conjugating enzymes of the ubiquitin system (Chen and Pickart, 1990). Incubations contained 50 mM Tris-Cl pH 8. 0, 2 mM ATP, 5 mM MgCl2, 5 mM phosphocreatine, 0. 3 units/ml phosphocreatine kinase, 0. 3 U/ml inorganic pyrophosphatase, 10 pg/ml ovalbumin, 30 uM E2-25k (plasmid obtained from Cecile Pickart), 0. 1 uM El from rabbit liver (A. L. Haas), and 5 to 10 mg/ml ubiquitin.

Reaction mixtures were incubated at 37°C for 40 min. The El and E2 enzymes were removed by passing the reaction mixture over a Mono Q anion exchange column (Pharmacia) at pH 7. 6. Polyubiquitin chains were purified by chromatography on Mono S FPLC (Pharmacia) as described above for proubiquitin. As used herein, the term"polyubiquitin chains"or"polymeric ubiquitin derivatives"are named as follows. The polyprotein ubiquitin gene product (UBI4p in yeast) is referred to as proubiquitin. The products of the UBI1, 2 and 3 genes in yeast are referred to as ubiquitin C-terminal extension proteins (UbCEP). The length of the CEP can be added as a suffix ; i. e. UbCEP52 or UbCEP76 in yeast. When the C-terminal carboxyl group of ubiquitin is involved in an amide bond, it is referred to as the ubiquitinyl group (Ub). The amino component of this amide bond can be contributed by either the amino terminus of a peptide (Na-ubiquitinyl-peptide) or the c-amino group of lysine (Ns-ubiquitinyl-lysine). Where known, the number of the specific lysine in a

peptide can be specified as a superscript prefix. Thus, a K48 linked ubiquitin dimer is referred to as Ns-ubiquitinyl-K48IUb. A larger polymer of £-linked ubiquitin is referred to as polyubiquitin, with the identity of the specific lysine involved specified as a superscript prefix (i. e. K48polyubiquitin, K63polyubiquitin, etc.).

Also, as used herein, the nomenclature referring to amino acids of the substrate, from the N-terminus, amino acids of the substrate are abbreviated as.... P3- P2-Pl-Pl'-P2'-P3'.... The scissile bond is that between the Pl and the Pl'residue.

The corresponding sites on the enzyme are labeled... S3-S2-Sl-Sl'-S2'-S3'etc. Other abbreviations are : UBP, ubiquitin-specific processing protease ; UCH, ubiquitin C-terminal hydrolase ; and SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

An s-linked ubiquitin dimer missing the C-terminal glycylglycine (Ne- ubiquitinyl-K48Ub 1'74) was synthesized as described above for NE-ubiquitinyl-K48b, except that 6 mg/ml of des-glygly-ubiquitin was reacted with 2 mg/ml of native ubiquitin. The reaction was incubated at 37°C overnight. Progress of the synthesis was assayed with HPLC, and terminated by the method outlined above. Under these conditions, polyubiquitin chains are <97% terminated with des-glygly-Ub. The reaction products were separated on Mono S FPLC (Pharmacia).

Ne-Ubiquitinyl-L-lysine and Ns-ubiquitinyl-K48Ub-L-lysine derivatives were synthesized as above except that the reactions included 200 mM to 500 mM concentration of the particular lysine derivative : either Na-acetyl-L-lysine (500 mM), Ne-acetyl-L-lysine (500 mM), L-lysine (200 mM), or Na-acetyl-L-lysine-N-methyl amide (200 mM). These reactions were allowed to incubate overnight at 37°C to assure maximal lysine conjugation. C-8 Reverse phase HPLC was used to monitor these reactions, and the reactions were terminated as described above.

Hydrolysis studies Hydrolysis rates were measured by incubating the above substrates with homogeneous UCH-L1 or L3. Conditions for assay were essentially as described previously (Wilkinson etal., 1986). Incubation of UCH was performed at 37°C in 50 mM Tris-Cl, pH 7. 6, with 5 mM DTT and 50 eug/ml ovalbumin, for varying amounts of time. Substrate concentrations were 15 J. M, approximately 20-fold higher than the Km for ubiquitin ethyl ester. Values are reported as the mean and the standard error of the mean for between 6 and 30 determinations. In cases where no catalysis was observed, the substrate was raised to its highest possible concentration.

Results Pl'specificity Removal of a single amino acid or small peptide from the C-terminus of ubiquitin must occur during processing of ubiquitin precursors and metabolites (see Table 4). As ubiquitin ethyl ester (Wilkinson et al., 1986) and Ub-DTT (Rose and Warms, 1983) are both rapidly hydrolyzed by UCH isozymes, it was of interest to determine if these enzymes exerted any specificity for residues at the Pl'position of ubiquitin fusion proteins2. Such specificity might manifest itself in differential rates of cleavage of a-linked amino acid extensions. FIG. 1 shows the hydrolysis rates obtained with UCH-L1 and-L3 isozymes for Ub-pro, Ub-lys, Ub-his, and Ub-asp, relative to ubiquitin ethyl ester, the inventors'generic reference substrate. The data show that neither UCH isozyme exhibited a strong preference for the P 1'residue (1) immediately following ubiquitin, except when it was proline. Ub-amino acid extensions were hydrolyzed by both UCH isozymes at rates only 1 to 2 orders of magnitude more slowly than UbOEt, whereas Ub-Pro was hydrolyzed at about 3 or 5 orders of magnitude more slowly than UbOEt (Table 3). These rates wqre determined at 15 uM substrates and probably represent Vmax values. Thus, these UCH isozymes are not selective with respect to the charge or size of residues at the PI'position when the ubiquitin extension is a single non-proline amino acid residue.

Table 3 : Rates of hydrolysis of ubiquitin derivatives by UCH-L1 and UCH-L3 Substrate UCH-L1 Activity UCH-L3 Activity (jumoles/min/mg) (unoles/min/mg) lMean 1SEM lMean 1SEM 1. Ub- [OEt] 30 6. 0 110 22 2. Na-Ub- [L-histidine] 6. 0 0. 9 26 2. 6 3. Na-Ub- [L-lysine] 7. 2 0. 7 20 4. 0 4. Ng-Ub-[L-lysine] 3. 7 1. 4 23 4. 0 5. Ns-Ub- [Na-acetyl-L-lysine] 6. 3 1. 3 13 2. 6 6. Na-(Ub-K48Ub)-[L-lysine] 4. 7 0. 3 9. 9 2. 2 7. N#-(Ub-K48Ub)-[Nα-acetyl-L- 5. 0 1. 0 10 2. 0 lysine] 8. Na-Ub- [L-aspartate] 9. 9x10-1 9.0x10-1 15 2. 4 9. Nα-Ub-[MQIFVRPR] 1. 5x10-1 6. 3 x10'2 79 8. 8 10. Na-Ub- [MQIFVKTLTG] 6. 0 x10-3 1. 9 x10-3 8. 8 1. 7 11. Nα-Ub-[HEPSLRQLA] 1. 4x10-4 6.6x10-5 8.5 0. 9 12. Nα-Ub-[CEP52] 2. 1x10-4 4.8x10-4 8. 4 5. 9 13. Na-Ub-[L-proline] 3. 9x10" 2. 5 x10-3 1. 3 x10-1 8. 8 X10-2 14. Nα-Ub-[UB] <1x10-5 - <1 x10-5 - 15. Nα-Ub-[PRSLDSC] <1x10-5 - <1x10-5 - Rates of hydrolysis of the indicated substrates are shown. The leaving group is bracketed. The detection limit in this assay is about 1#10-5 µmoles/min/mg. 1The mean and the standard error of the mean were derived from between 6 and 30 replicate measurements.

Table 4 : Illustrative C-terminal Extensions of the Proubiquitin Gene Product in Various Organisms Extension Organism (# Ub repeats) -AF Acetabularia cliftonii (9) -C Bos taurus (4), Homo sapiens (3) -DI Caenorhabditis elegans (11) -DF Petroselinum crispum (6) -F Geodia cyndonium (6), Nicotiniana sylvestris (6), Pisum sativum (5), Arabidopsis thaliana (5), Glycine max (4), Antirhinium majus (>3), Sus scrofa (>3), Candida albicans (3), Euplotes eurystomus (3), -IQA Drosophila melanogaster (3) -K Hordeum vulgare (>2) <BR> <BR> <BR> <BR> -L Dictyostelium discoideum (5 and 3), Trypanosoma brucei<BR> <BR> <BR> <BR> <BR> <BR> <BR> brucei (I) -M Aglaothamnion neglectum (6)

Table 4 (continued) -N Dictyostelium discoideum (7 and 5), Gallus gallus (3), Phytophtora infestans (3) -Q Strongylocentrotus purpuratus (10), Zea mays (7), Oryza sativa (6), Tetrahymena pyriformis (5), Avena fatua (4), Neurospora crassa (4) -TQTSGKTFMTELTL Artemius nauplius (>2) -VYASPIF Cavia porcellus (4) -V Homo sapiens (9) -Y Cricetulus griseus (5), Gallus gallus (4), Mus musculus (4) The proubiquitin genes of most organisms encode head-to-tail repeats of the ubiquitin coding sequence with an additional amino acid or peptide at the C-terminus.

A wide variety of residues must be cleaved from the polyubiquitin gene containing a variable number of ubiquitin repeats. Hence, the activity responsible for this cleavage are expected to show little P I'specificity. Note the absence of proline at the junction.

Comparison of peptidase and isopeptidase activities Because ubiquitin is also conjugated to proteins through an isopeptide bond (i. e. through the s-amino group of lysine), it was of interest to examine whether UCH isozymes could cleave Ub-E-amino lysine derivatives. It has been shown that UCH- L3 can hydrolyze both types of bonds (Pickart and Rose, 1985), although absolute rates were not determined. As a model isopeptidase substrate, the inventors synthesized Ns-ubiquitinyl lysine by incubation of ubiquitin and lysine with the El activating enzyme, the E2-25K conjugating enzyme and ATP. In this synthesis, the excess lysine nucleophile captures the thiolesterified ubiquitin from the transient E2- Ub intermediate, forming exclusively the Ns-ubiquitinyl lysine product and halting further synthesis of polyubiquitin by E2-25k. Both UCH isozymes rapidly hydrolyzed Ne-linked lysine (FIG. 10). Additionally, the rates were essentially identical to those obtained with Na-linked lysine (Table 2).

Table 2. Refinement Statistics Resolution Range (A) 6. 0-1. 8 High resolution shell (A) (1. 88-1. 80) Rvalue (%) a 23. 0 (36. 9) Rfree (%) b 28. 6 (36. 4) rmsd (bonds) (Å) c 0. 0l0 rmsd (angles) (O) C 1. 867 #Residues included (total) 205 (230) #Atoms with occupancy-0. Od 10 #Water molecules 121 <B> (Å2) Protein/Water 27. 9/36. 6 # (p angles (%) : Most Favored 92. 8 Additional 6. 7 Generous 0. 6 Forbidden 0 Rvalue = 100 * E Fp (obs) # - #Fp (calc) # = ##Fp (obs) # b Rfree = Rvalue for a randomly selected subset (5%) of the data that were not used for minimization of the crystallographic residual (Brünger, 1992a). c Stereochemistry was analyzed with PROCHECK (Laskowski et al., 1993). d Non-hydrogen atoms only. Atoms of the Arg-145 and Glu-203 side chains were assigned an occupancy of zero because they lack defined electron density.

A more relevant e-linked substrate might be an NE-ubiquitinated peptide similar to the degradation remnants expected to be generated by the action of the proteasome on ubiquitinated proteins. To more closely mimic a peptide bond at the a amino group of an Ne-linked lysine, the inventors synthesized and tested Ne-

ubiquitinyl- (N-a-acetyl) lysine as a substrate. The addition of an acetyl functionality to the a-amino group did not affect the hydrolysis rate of Ne-ubiquitinyl-lysine (FIG. 10). Both isozymes cleaved acetylated and unacetylated substrates at a rate roughly 8 to 10 fold slower than the rate of cleavage of ubiquitin ethyl ester.

Subsequent studies showed that there was also no effect of amidating the carboxyl group of lysine with N-methyl amine.

Polyubiquitin processing Because Ns-ubiquitinyl-lysine was a good UCH substrate, the inventors sought to determine if an NE-diubiquitinyl lysine derivatives were good UCH substrate. If these enzymes function in the removal of a K48-linked remnant peptide from polyubiquitin, they should process lysine derivatives at the C-terminus of polyubiquitin chains. As a model substrate, the inventors synthesized Nc- (Ub- Ub)-lysine and Ns-(Ub-K48Ub)-(N-a-acetyl) lysine. The lysine is removed from these polyubiquitin derivatives at rates identical to the simpler Ne-Ub-lysine derivatives, regardless of the presence of a second ubiquitin (FIG. 1). Neither UCH is able to hydrolyze the K48 isopeptide bond. Neither Ub-K48Ub, nor Ub-K4SUb (des- glygly) is cleaved, even at a four-fold molar excess of enzyme for two hours at 37°C.

This hydrolysis rate is therefore more than eight orders of magnitude slower than the ubiquitin esterase rate of either enzyme. This suggests that the ubiquitin binding site on UCH isozymes recognizes a face of ubiquitin distant from the K48 linkage site, and suggests that UCH could function in generating a free C-terminus on polyubiquitin chains by the removal of small peptides and/or cellular nucleophiles.

Fusion peptide processing It has been postulated that Ubiquitin C-terminal hydrolases could participate in the processing of ubiquitin gene products. It is unlikely that a protein as small as UCH could exhibit specificity for ubiquitin and also a significant portion of the C-terminal extension. Thus, if UCH activity were responsible for processing

ubiquitin gene products, then these enzymes would be expected to exhibit specificity for the peptide sequences at the junction between ubiquitin and the C-terminal extension. Model substrates synthesized to test this hypothesis consisted of ubiquitin followed by the first ten amino acids of the C-terminal extensions ; i. e., Ub-CEP521-o (substrate 11, FIG. 10) and Ub-Ubl-lo (substrate 10, FIG. 10).

FIG. 10 shows that isozyme L3 exhibited little selectivity for any of the peptide extensions, cleaving them nearly as rapidly as it cleaves single amino acid extensions. This is also consistent with data which suggests that UCH-L3 has no difficulty cleaving a wide variety of peptide substrates from ubiquitin if the peptides are less than about twenty residues. Interestingly, UCH-Ll exhibited considerably more specificity, showing rates of hydrolysis of these substrates that are over two orders of magnitude slower than the rates of L3-catalyzed hydrolysis (Table 3). Still, UCH-Ll exhibits notable selectivity ; the Ub-Ub-l° substrate is hydrolyzed over forty fold faster than the Ub-CEPI-O substrates by this enzyme.

Aside from the natural peptide sequences at the C-terminus of ubiquitin, one other substrate was created. Ub-PRSLDSC, a ubiquitin-peptide fusion with proline at the P'cleavage junction was created by a PCRTM error that resulted in the read- through of the reading frame into the vector multicloning site. Neither enzyme was able to cleave this fusion peptide at a measurable rate, in spite of the fact that UCH-L3 is able to cleave Ub-pro. The hydrolysis rate of these peptide fusions was more than seven orders of magnitude slower than that for UbOEt.

Ubiquitin proprotein processing Because model substrates containing the first ten residues of ubiquitin proproteins were hydrolyzed by UCH isozymes, the inventors determined the rate of cleavage of full-length ubiquitin gene products by these enzymes. Purified a-linked Ub oligomers were very slow substrates for UCH-L1, and were not cleaved at all by UCH-L3 (FIG. 10). Micromolar UCH-L1 was able to cleave Na-diubiquitin at 37°C

in vitro with a half-life of thirty minutes. This corresponds to a rate of at least 6 orders of magnitude slower than for UbOEt. UCH-L1 is reported to exist at 1-2 % of total soluble brain protein (Day, 1990).

The zinC-finger fusion proteins UbCEP52 and UbCEP80 are the two other natural ubiquitin proprotein substrates studied. High amounts (100 mU) of either recombinant UCH added to bacterial expression lysates for two hours failed to hydrolyze UbCEP52 or UbCEP80 to their monomeric components, based on immunoblotting of the expression lysates. Because UbCEP52 was more highly expressed than UbCEP80, and because the antibodies to CEP52 had a higher titer and were more specific than the anti-CEP80 antibodies, the UCH-catalyzed hydrolysis of the UbCEP52 protein was further characterized.

Surprisingly, purified UbCEP52 was hydrolyzed by both enzymes, though the L3 isozyme catalyzed the reaction much more rapidly (FIG. 10 and FIG. 11). The rate of processing of UbCEP52 by UCH-L3 approaches the rate of hydrolysis of the Ub- amino acid extensions, about 200 mirai 1. To confirm the specificity of this reaction, SDS-PAGE and immunoblotting were used to identify the products (FIG. 11. The appearance of ubiquitin and CEP52 detected by SDS-PAGE is consistent with the rates measured by HPLC. UCH-L1 also hydrolyzed the substrate to a measurable degree, but the rate was 2. 1 x 10 gmoles/min/mg, or about 10-5 the rate of ester hydrolysis.

The above results suggest that the bacterial lysates contain something which interferes with UbCEP52 hydrolysis, but not with UbOEt hydrolysis. UbCEP52 possesses a C2H2 zinC-finger binding motif, so it was determined whether binding of zinc could inhibit the UCH-L3 hydrolysis of UbCEP52. Zn (OAc) 2 (10 mM) did not inhibit UbCEP52 hydrolysis by either enzyme. Whether the zinC-finger motif binds metal in vivo remains to be elucidated, however, addition of excess metal ion does not inhibit the processing of the proprotein by UCH.

The presence of a zinc finger motif in a ribosomal protein is presumptive evidence of nucleic acid binding. To test if binding of nucleic acid inhibited processing, assays were performed in the presence of nucleic acids. In vitro addition of 50 pg/ml of either plasmid DNA, or a double stranded 26-base pair DNA cassette inhibited the hydrolysis of UbCEP52 by 50%, whereas a single stranded 42-base pair oligodeoxynucleotide at the same concentration was only minimally effective (FIG. 12). Whole yeast RNA was even better at inhibiting processing, showing 60 to 80% inhibition. Phenol/chloroform extraction of this RNA did not improve the processing, suggesting that the inhibition was not due to other contaminating proteins in the RNA preparation. Also, preincubation of the RNA with RNAseA restored the UbCEP hydrolysis rate back to control rates. These results imply that the nascent proprotein can only be cleaved by UCH before nucleic acids are bound to the fusion peptide, and that assembly into the ribosomal subunit would probably prevent processing.

UCH isozymes can co-translationally process ubiquitin proproteins.

Ubiquitin proproteins are very rapidly processed in vivo (Finley etal., 1989 ; Baker etal., 1992). The UCH isozymes appear to be very efficient at processing peptides from the C-terminus of ubiquitin, but not if the C-terminal extension has a chance to fold into a tight, globular domain (see above). Further, only UCH-L1 is able to rapidly process the proubiquitin precursor, and this isozyme is present at low levels in most tissues. These observations suggest that processing of some ubiquitin gene products may occur before folding or subunit assembly is completed. To test the idea that UCH isozymes can process UbCEPs co-translationally, the inventors co- transformed cells with vectors expressing UCH and Ub proproteins in various combinations. UCH-L1 was found to hydrolyze polyubiquitin (60%) and UbCEP80 (50%), but not the UbCEP52 (>5%) (FIG. 13). This data is consistent with the above data from peptide hydrolysis, in that UCH-L1 prefers to hydrolyze ubiquitin-like peptides and also hydrolyzes the complete proubiquitin, albeit slowly. In contrast,

UCH-L3 was found to hydrolyze both Ub-CEP fusions, but not proubiquitin (FIG. 13).

The present invention describes attributes relating to the substrate specificity of two closely related UCH isozymes, UCH-L1 and-L3. The hydrolysis rates reported herein were determined at 15 uM substrates, approximately the same concentration as that of total ubiquitin in the cell. The Km for hydrolysis of ubiquitin ethyl ester is approximately 1 uM and is identical to the ubiquitin binding constant (Larsen et al., 1996). Thus, in the absence of unfavorable interactions between the enzyme and the leaving groups, the measured rates would reflect Vmax values. With some of the poorer substrates however, the slower observed rates of hydrolysis may be due to higher Km values for these substrates. Irrespective of the reasons for the slower rates of hydrolysis, it is clear that these differences are manifest at concentrations that are many times that observed in a cell and that the rates reported may overestimate the relative rates of hydrolysis that would pertain in vivo.

Ubiquitin binding to the S site (s) The available evidence suggests that the S sites form an extensive binding site for intact ubiquitin. The only demonstrated activity of UCH isozymes is for cleavage of amide and ester bonds at the C-terminus of ubiquitin. There is little or no affinity for small peptides at the C-terminus of ubiquitin (such as glycylglycine) but ubiquitin is bound with a micromolar binding constant (Larsen et al., 1996). Ubiquitin aldehyde is a tightly-bound inhibitor of these enzymes. Further, NMR measurements have confirmed an extensive area of contact between ubiquitin and UCH-L3 ; encompassing over 20% of the surface residues on ubiquitin (Wand and Wilkinson) including the C-terminus. This contact surface cannot include the N-terminus of ubiquitin, as a hexahistidine tag at the N-terminus has little or no effect on the rates of hydrolysis. In agreement with this result, it has been shown that these enzymes bind to immobilized (his) 6 ubiquitin (Beers and Callis, 1993). The surface of ubiquitin

containing K48 is also not in the Sl recognition site on ubiquitin, as N£-Ub-K48Ub derivatives are good substrates for cleavage of the leaving group from the free C-terminus (FIG. 10 and Table 3). Ne-Ub-K48Ub does not appear to be a substrate, probably because the leaving group ubiquitin is tightly folded against the C-terminal face of the distal ubiquitin. Finally, the interactions between ubiquitin and UCH-L3 are predominantly ionic, as evidenced by the previously observed inhibition of binding and activity by salt (Larsen et al., 1996).

Sl'Specificity Many different amino acids and peptides are found as natural extensions of ubiquitin genes in eukaryotes (Table 4). Putative processing enzymes would have to either have broad specificity at the Pl'site or exhibit significant sequence variability from species to species in order to accommodate their respective species-specific leaving groups. In fact, UCH sequences are very similar across species, with rat, human and bovine UCH-Ll being over 98% identical. The inventors'results show that UCH isozymes exhibit very little specificity for the Pl'residue of ubiquitin substrates (FIG. 10) with essentially identical rates with acidic, basic or neutral leaving groups. If UCH isozymes were responsible for processing the amino acid extensions of ubiquitin gene products, they would exert little selective pressure on the nature of that leaving group. This may be why there seems to be little selective pressure to maintain the identity of this extension amino acid (see Table 4).

As both a-, and £-linked derivatives have to be processed from the C-terminus of ubiquitin, the selectivity for cleavage of these two types of amide bonds was examined. These enzymes exhibited little or no discrimination based on the identity of the amide bond to lysine (a vs. s), the charge at the other amine (free amine vs. N- acetyl), or the charge at the carboxyl group (carboxyl vs. N-methyl amide). Further, the same derivatives can be efficiently processed from the C-terminus of the polyubiquitin chain. Thus, at least with small leaving groups, these enzymes could be

involved in processing both the amino acid and small peptide extensions of various gene products, as well as the Ns- (poly) ubiquitinyl lysine expected to be generated by the action of the proteasome on polyubiquitinated protein substrates.

The S site (s) Will Not Bind Larger Protein Domains FIG. 10 demonstrates that UCH-L3 is generally able to hydrolyze a variety of small peptide fusions at the C-terminus of ubiquitin. To examine if there was any selectivity based upon P'sequences, the inventors have also measured the rates of processing of the ubiquitin gene products and short model substrates consisting of ubiquitin fused to the first ten amino acids of the C-terminal domains.

UCH isozymes exhibit significant selectivity in the processing of the ubiquitin gene products. UCH-L3 is able to efficiently process the Ub-CEP52 gene product, but not the Ub-CEP80 or proubiquitin gene products. Isozyme L1 is only able to slowly process the proubiquitin gene product in vitro and in vivo. It has been reported that the yeast homolog, YUHl, also exhibits a similar selectivity in that small fusion proteins can be efficiently processed, but not larger fusions (Miller et al., 1989). The drosophila homolog has been reported to be able to process a-ubiquitinyl-IKBa (314 amino acids), but not larger fusions (Roff et al., 1996).

Interestingly, nucleic acid binding to Ub-CEP52 likely prevented its processing by UCH-L3 (FIG. 12). The addition of nucleic acid to UbOEt had no effect on its hydrolysis, suggesting that the nucleic acid was directly binding to Ub- CEP52 and causing a conformational change which prevented processing. The binding of nucleic acid by Ub-CEP52 is not unexpected ; the CEP domain contains a zinC-finger motif, the protein is a ribosomal subunit, and mutants in this gene are defective in rRNA processing.

The above results suggest that the selectivity of the S'sites may be based on factors other than size. One factor could be the accessibility of the peptide bond at the

C-terminus of ubiquitin. It might be expected that ubiquitin fusion proteins with significant mobility and flexibility at the junction could be good substrates while those that are more constrained (proline) and/or sterically restricted (large) would be poor substrates. This is consistent with the ligand-induced inhibition described above (i. e. binding of nucleic acid may cause a less mobile conformation around the Ub- CEP52 junction) as well as the restricted nature of the substrate binding cleft observed in the UCH-L3 crystal structure (Johnston et al., 1997).

Substrate Specificity Based on the P'Peptide Sequence An alternative explanation for the observed selectivity in processing of ubiquitin gene products is that the enzymes may exhibit significant selectivity based on the amino acid sequences binding to the S'sites. To examine the contribution of the P'residues to the observed selectivity, the inventors have used model substrates consisting of ubiquitin fused to small peptides, including the first ten amino acids of each ubiquitin gene product. FIG. 10 demonstrates that UCH-L3 is not very selective for the P'residues, processing every small peptide tested except those containing proline at the scissile bond. This specificity is similar to that reported for the yeast UCH ; i. e. ubiquitin extended by E, C, D, G, T, or M (but not P) was hydrolyzed efficiently (Miller etal., 1989). This may be because the secondary amine of the proline has a somewhat higher pKa than the primary amino group in the peptide bond of most amino acids, or it may reflect a steric constraint imposed at the scissile bond.

UCH-L3 is unable to process at proline in the Ub-PRSLDSC peptide fusion. It is likely that the presence of proline at the P I'position"kinks"the peptide such that it can not be accommodated in the active site cleft. The presence of a proline at position P4' (Ub-CEP521uo, substrate 11) or P7' (Ub-Ubl-5-RPR, substrate 9) has little effect on the rate of peptide processing by UCH-L3, suggesting that the cleft may be considerably less restricted at that distance from the active site nucleophile. The Ub- Ubl-l° construct is processed very effectively by UCH-L3, but the Ub-Ub fusion

protein is not cleaved at all, reinforcing the conclusion that a tightly folded domain at the C-terminus of ubiquitin is not generally a substrate for these enzymes.

In contrast to the permissiveness of UCH-L3 processing, the processing by UCH-L1 is more selective, with ubiquitin related peptide fusions being reasonable substrates and Ub-CEP52 1-10 being a poor substrate. While it is not clear whether this selectivity is due to subsite specificity at P1'-P3', or the presence of proline in sites P4'-P7', it is clear that this is a much more selective enzyme. This specificity may be related to interactions with an occluding loop which is postulated to form part of the S'sites on the UCH family of enzymes.

Co-translational Processing These results demonstrate that there is considerable selectivity in the processing of ubiquitin gene products by these UCH isozymes. UCH-L3 appears to prefer processing of Ub-CEP gene products, while UCH-L1 is very selective for the proubiquitin gene product. There is, however, some question as to the physiological significance of these processing events, especially those catalyzed by UCH-L1 which occur at an extremely slow rate. This led the inventors to ask if these enzymes might be involved in co-translational processing. Normal processing is known to be extremely efficient, with no evidence for accumulation of intermediates in the process.

Further, if these enzymes are involved in processing, they must act before significant assembly into ribosomal subunits, and/or folding of stable domains C-terminal to ubiquitin. When enzyme and substrate were co-expressed in E. coli, the efficiency of processing was high and the selectivity was similar to that observed above. UCH-L1 was able to process over 80% of the proubiquitin gene product, and little of the Ub- CEP gene products, while UCH-L3 was most efficient in processing the Ub-CEP fusion proteins (> 50% processed). Thus, it appears that processing is much more efficient if the enzyme is present during the synthesis of the substrate. Confirmation of this phenomenon was attempted by demonstrating the association of UCH-L3 with polyribosomes synthesizing the substrates. When an in vitro transcription/translation

system is supplemented with DNA encoding the substrate, endogenous UCH activity is found exclusively in the soluble fractions. Even upon addition of exogenous UCH isozymes, little or no UCH activity can be found stably associated with the ribosomes.

It may be that the association is only fleeting and unstable, or it may be that processing occurs after release of the substrate polyprotein from the ribosome but before folding of the complete protein.

Molecular Basis of Specificity As shown above, the x-ray crystal structure of UCH-L3 has a core catalytic structure that strongly resembles cathepsin B, a papain-like protease. The active site groove is occluded by two loops, and it is postulated that a substrate-induced conformational change is required to clear the cleft and allow access to the active-site cysteine. Thus, only ubiquitin derivatives are substrates because only they can form the extensive interactions with the S'site required to trigger the necessary conformational change generating the active conformation of the enzyme.

Specificity for P'residues must be determined by the residues lining the corresponding S'sites on the UCH enzymes. The sequence of these proteins varies widely in several areas, including a region just N-terminal to the active site histidine.

This sequence is disordered in the UCH-L3 structure, but may be positioned to form a significant contact region with the P'residues of substrates (Johnston et al., 1997).

Thus, it is likely that this hypervariable region is important in determining substrate selectivity and the somewhat shorter loop near the active site cysteine in UCH-Ll restricts the possible substrates by conferring a narrower or more restricted active site cleft. These predictions could be tested by obtaining the structure of UCH-L 1 and/or using site directed mutagenesis and domain swapping approaches.

Potential Physiological Roles for UCH Isozymes The possible physiological roles for UCH isozymes are limited by the temporal and spatial patterns of expression of the enzymes and putative substrates, as well as by restrictions imposed by the substrate specificity examined here. With respect to the former, there is a marked tissue specificity to the expression of UCH isozymes, with UCH-L1 being expressed at very high levels in neural and diffuse neuroendocrine tissues, and UCH-L3 being expressed primarily in hematopoetic tissues (Wilkinson et aL, 1992). There is little evidence of temporal regulation, as these enzymes seem to be present in all stages of the cell cycle and both early and late in development. A third isozyme, UCH-L2 has been reported to be widely distributed, albeit at lower levels than either of the two isozymes studied here (Wilkinson et al., 1992).

The distribution of putative substrates is more difficult to assess, although the results discussed above suggest that substrates will include the ubiquitin proproteins and small molecule adducts of ubiquitin. The latter are expected to be widely distributed, as there is extensive activation and conjugation of ubiquitin in all tissues examined. All of the intermediates in the enzymatic activation of the C-terminus of ubiquitin are thiol esters and they are effectively trapped by reaction with small molecular weight thiols and amines. There is a much more specific expression of ubiquitin pro-proteins. Rapidly growing cells have been shown to express high levels of ubiquitin-ribosomal fusion proteins, while more differentiated cells (such as neurons), express ubiquitin primarily from the proubiquitin locus.

These considerations suggest that UCH-L1, the neuronal specific isozyme, may be more efficient at cleaving the proubiquitin precursor, while the hematopoetic specific UCH-L3 might prefer ubiquitin ribosomal fusion proteins as substrates.

These predictions are borne out using ubiquitin fusion peptides as substrates. UCH- Ll is found at high levels only in neurons and diffuse neuroendocrine tissues, and it

cleaves the proubiquitin model substrate (Ub-Ubl-l0, substrate 10) much faster than it cleaves the ubiquitin ribosomal fusion protein model substrate Ub-CEP52 1-10 (substrate 11). UCH-L3 on the other hand can cleave all the model substrates at a significant rate. The specificity of co-translational cleavage of the full length gene products reflects the results with small peptide fusions, implying that a portion of the UCH specificity derives from interactions with P'residues. Large, tightly folded leaving groups are not substrates for this class of enzyme, although there are differences in the selectivity demonstrated by each enzyme.

These results support the idea that UCH enzymes are responsible for co- translational processing of the polymeric ubiquitin gene products and/or salvage of ubiquitin from small molecular weight adducts. Only ubiquitin derivatives will be substrates, probably because of the obligatory substrate-induced conformational change required to generate the active enzyme. Isozymic differences may be due to sequence differences in the hypervariable loop region and presumably reflect the metabolic flux of the tissues wherein these isozymes are expressed, although confirmation of this role awaits identification of mutations in these loci or development of transgenic animal models.

EXAMPLE 3-SUBSTRATE BINDING AND CATALYSIS BY UBIOUITIN C-TERMINAL HYDROLASES There are several polymeric ubiquitin structures which contribute to the biology of ubiquitin. Ubiquitin is post-translationally conjugated to a variety of proteins present in the cell. Proteins can be multiubiquitinated by the addition of ubiquitin to several surface lysines or polyubiquitinated by the addition of ubiquitin to one surface lysine followed by the addition of another ubiquitin to K48 of the first ubiquitin. Long polymeric chains can thus be assembled by the conjugation of ubiquitin to the distal end of this chain. These polyubiquitinated proteins are then degraded by the 26S proteasome to yield free amino acids and the polyubiquitin chain (Eytan et al., 1989 ; Hough et al., 1987). The ubiquitin isopeptide bond linking these

subunits must be hydrolyzed by the action of specific proteases. This hydrolysis is necessary to salvage ubiquitin for conjugation as well as to prevent the accumulation of free polyubiquitin chains which are known to bind to the 26S proteasome and inhibit proteolysis (Deveraux et al., 1994). The inventors have recently shown that this reaction is catalyzed by a 93 kDa protein termed isopeptidase T (Wilkinson et al., 1995).

In addition to isopeptide-linked polymeric ubiquitin, the cell must also proteolytically process polymeric ubiquitin linked by peptide bonds. Ubiquitin is always translated from mRNA as a fusion protein, either with additional copies of ubiquitin itself or with one of two different zinc fingers (Ozkaynak et al., 1987). The proubiquitin gene product consists of multiple copies of ubiquitin, is induced by stress, and must be processed to monomeric ubiquitin by the action of a processing protease (Finley et al., 1987. Similarly, two ubiquitin-zinc finger fusion proteins arc synthesized in rapidly growing cells. They must be accurately processed to free ubiquitin and the zinc finger CEP52 arid CEP80, which are ribosomal proteins (Finley et al. 1989).

The proteolytic processing of both a-and s-amide linked ubiquitin occurs at the carboxyl group of glycine 76, suggesting that such processing proteases might have specificity for binding the ubiquitin monomer. Several proteases with these properties have been described, including those known as ubiquitin C-terminal hydrolases (Pickart and Rose, 1985), ubiquitin specific proteases (Tobias and Varshavsky, 1991 ; Baker et al., 1992), or isopeptidases (Matsui et al., 1982). These proteases can be grouped into two families. The ubiquitin-specific protease family (UBP) consists of several distantly-related proteases of 50-300 kDa which show several homologies around an active site thiol and a putative active site histidine.

(Abbreviations used : CD, circular dichroism ; DTT, DL-dithiothreitol ; EDTA, ethylenediaminetetraacetic acid ; IPTG, isopropyl ß-D-thiogalactopyranoside ; MES, 2-[N-morpholino] ethanesulfonic acid ; PAGE, polyacrylamide gel electrophoresis ;

PCRTM, polymerase chain reaction ; PMSF, phenylmethylsulfonyl fluoride ; SDS, sodium dodecyl sulfate ; Tris, tris (hydroxymethyl) aminomethane ; Ub, ubiquitin ; UbOEt, ubiquitin ethyl ester (Wilkinson et al., 1986) ; UBP, ubiquitin specific proteases (Baker et al., 1992) ; UCH, ubiquitin carboxyl-terminal hydrolase (Wilkinson et al., 1989). This family is also known as UCH family 2 and includes at least 11 members in yeast with other known homologues in mammals and Drosophila (Papa and Hochstrasser, 1993 ; Wilkinson et al., 1995). They are thought to be involved with processing various ubiquitin-protein fusions expressed in eukaryotic cells and/or the polyubiquitin degradation signal (Tobias and Varshavsky, 1991 ; Baker et al., 1992, Wilkinson et al., 1995). The ubiquitin carboxyl-terminal hydrolase (UCH) family is a group of small, closely-related thiol proteases consisting of three mammalian isozymes (Wilkinson et al., 1989) and with close homologues in Saccharomyces cerevisae (Liu et al., 1989) and Drosophila melanogaster (Zhang et al., 1993). They exhibit no apparent homology to the UBP family, and this dissimilarity implies two functionally convergent ancestral genes. The presence of multiple, tissue specific UCH isozymes (Wilkinson et al., 1992) suggests that the metabolism of ubiquitin may also be tissue specific. These enzymes prefer small leaving groups and/or extended peptide chains at the C-terminus of ubiquitin. It is postulated that they are involved in the co-translational processing of the proubiquitin and ubiquitin-zinc finger fusion proteins which are the ubiquitin gene products. It is not clear how any of these processing proteases distinguish among the several types of polymeric ubiquitin or achieve hydrolytic specificity. Since many, if not all of them, bind ubiquitin, their hydrolytic specificities and in vivo rates may depend on the specific recognition of leaving group peptides, side chains, or proteins in the non- ubiquitin portion of the substrate the P'site according to the nomenclature of Schechter and Berger (1967).

The UCH class of proteases is unique in several ways. Firstly, they appear to represent a new family of thiol proteases, as there is no apparent sequence homology to any other proteases. As such, the structure and function of these proteins is of

general interest. Secondly, they are extremely specific, cleaving only after the C-terminal glycine of ubiquitin. Recombinant UCH's can be expressed in high amounts in Escherichia coli, do not form inclusion bodies, and are nontoxic to the host. This is consistent with the enzymes having a very narrow spectrum of proteolytic specificity. In contrast with members of the papain super-family, which exhibit broad P site specificity (Fox et al., 1995), UCH's show strict and narrow P site specificities for the RGG C-terminus of Ub. Finally, these enzymes are mechanistically unique in that binding of ubiquitin results in a finite equilibrium of thiol ester between the C-terminus and the active site thiol of the protease. Thus, the enzyme-substrate complex (ubiquitin + UCH-L3) can be reduced by borohydride to give the thiohemiacetal of the protease and ubiquitin aldehyde (Pickart and Rose, 1986). The energy required to form even a small amount of intermediate thiol ester must result from extensive binding interactions between ubiquitin and the protease.

For these reasons, a more detailed structural analysis of the UCH family is of interest.

The inventors previously reported four UCH activities from bovine thymus with specificity for cleavage of the C-terminal ethyl ester of ubiquitin (Mayer and Wilkinson, 1989). Three of these enzymes are approximately 25 kDa in size, while the fourth activity is of higher molecular weight and is less well understood. These -25 kDa activities are named UCH-L1, UCH-L3 and UCH-L3 on the basis of their order of elution from a DE-52 anion exchange matrix, and the inventors have found UCH-L1 to be identical to the protein PGP 9. 5 (Wilkinson et al.. 1989). This hydrolase is most highly expressed in neuronal and neurosecretory tissues. Additionally, it is selectively accumulated (along with ubiquitin conjugates) in the plaques of Alzheimer's disease as well as in lesions of other neurodegenerative diseases (Lowe et al., 1990). In the present work, UCH-L1 was cloned and mutagenized, and three important residues were identified, including the active site cysteine and histidine. Various spectral characterizations demonstrate that UCH contains a/p folding motifs and that the UCH mutants studied demonstrate normal parameters of thermal denaturation. Thus, these residues appear to be unimportant for

protein folding or stability. As UCH-L1 is insoluble above 1. 5 mg/mL, the physical characteristics of a more tractable isozyme, UCH-L3, were studied. The inventors find that ubiquitin binding to this isozyme is stoichiometric and inhibited by salt.

These data provide the first detailed analysis of the binding of ubiquitin with one of its adjunct enzymes, and so provides additional insights into the nature of the ubiquitin UCH protein-protein binding interactions.

PROCEDURES UCH Cloning and Subcloning. The cDNA encoding UCH-L3 from the plasmid pBHA (Wilkinson et al., 1989) was subcloned into the T7 expression vector pRSET (Invitrogen, San Diego, CA). Plasmids pBHA and pRSET were digested with NdeI and EcoRI (New England Biolabs, Beverly, MA). The 780 bp UCH-3 insert and the 2810 bp vector were gel-purified and ligated, and the resultant plasmid was used to transform Top 10 F'E. coli (Invitrogen). Colony minipreps were screened, and several which were linearized by NdeI to give a 3. 5 kb linear fragment were selected.

An insert from a positive clone was sequenced to verify the integrity of the plasmid ("pRS-UCHL3") and was used to transform the E. coli expression host BL21 (DE3) (Novagen). On IPTG induction, cells with this plasmid overexpressed a 25 kDa protein which cross-reacted with anti-human UCH-L3 polyclonal antibodies. Cytosol from the sonicated cells showed significant enzymatic activity in cleaving ubiquitin ethyl ester (Wilkinson et al., 1986). Human UCH-L1 was cloned via reverse transcriptase-mediated polymerase chain reaction (RT-PCRTM, Perkin-Elmer Cetus) from a human fetal brain poly-A RNA library (obtained from Dr. Stephen T. Warren) using primers to the known human PGP9. 5 sequence (Day et al., 1990). It was subcloned by the dideoxy method (Sanger et al., 1977), subcloned into pRSET to give pRSLl, and transformed into BL21 E. coli as described above. Sequencing revealed two apparent PCRTM errors affecting the codons for residues 73 and 200. Since the change at codon 200 was silent, it was not corrected. The codon at position 73 was repaired as follows. A rat PGP9. 5 (UCH-L1) fragment (Kajimoto et al., 1992) was amplified by PCRTM to generate a new silent 5'BssHII site. The resulting

BssHII/DraIII cassette codes for identical residues in the rat and the human sequences and so was inserted into pRSLl in place of the human gene fragment. The construct was sequenced and shown to have the correct predicted amino acid sequence.

UCH Purification. The inventors cloned, expressed, and purified recombinant UCH-L1 and UCH-L3 to study their physical and enzymatic properties. With the exceptions noted, the purification of all UCH isozymes and mutants was similar. A single colony of BL21 (DE3) carrying the pRSET-UCH L3 plasmid was inoculated into 2 L of LB media (Sambrook et al., 1986) and grown at 37°C to an absorbance of 0. 8 at 600 nm. IPTG (Sigma, St. Louis, MO) was added at 0. 4 mM, and the cells were incubated for an additional 1. 5 h before the bacteria were centrifuged at 4000g and the pellets were collected. After induction, UCH levels reached an average of 15% of the soluble E. coli protein. The cell paste (16 g) was resuspended in 100 mL of lysis buffer (50 mM Tris-HCl, pH 7. 5, 10 mM DTT, 50 uM PMSF, 1 mM EDTA, 10 mM MgCl2). Lysozyme was added to 10 000 units/mL for 30 min, and the suspension was sonicated (Heat Systems, Inc.). The debris was removed by centrifugation at 10 000g for 40 min. The supernatant was concentrated to 50 mL by ultrafiltration (Amicon, YM-10) and applied to a 200 mL Fast Flow Q-Sepharose column equilibrated with buffer A (50 mM Tris. HCl, pH 7. 6 ; 0. 5 mM EDTA ; 5 mM DTT). The column was eluted with a 300 mL linear gradient to 0. 5 M NaCl in buffer A. Fractions with ubiquitin esterase activity eluted at 265 mM NaCl and contained the 25 kDa protein as determined on SDS-PAGE. Enzymatically active fractions from ion exchange were pooled and concentrated to 30 mL and applied to a 1 L Sephadex G-100 Superfine gel filtration column (Pharmacia) in buffer A. Active fractions were pooled again and shown to be >98% pure by Coomassie-stained SDS- PAGE. These detailed enzymes have been used for kinetic studies and for the CD and UV spectroscopy, but for Raman spectra the enzymes were further purified on Mono Q FPLC anion exchange, using the same buffers and gradient as in the ion exchange step described above. The homogeneous fractions were pooled and concentrated by ultrafiltration. Purifications of UCH-L 1 were similar to that for UCH-L3, except that

the anion exchange salt gradients were 1-300 mM NaCl, with UCH-L1 eluting at 110 mM. Homogeneous UCH-L 1 is obtained in two steps, due to slightly higher expression levels and weaker binding to Q-Sepharose. The inventors find the specific activities of homogeneous recombinant UCH-L1 and UCH-L3 are 30 and 110 pmol/min/mg, respectively, using ubiquitin ethyl ester as the substrate. By comparison, UCH-L1 from bovine brain has a specific activity of 25/@ « tmol/min/mg, and UCH-L3 purified from calf thymus exhibits a specific activity of approximately half the recombinant value. These enzymes are therefore fully active and has been shown to bind one mole of substrate per mole of enzyme (see below), suggesting that they are fully functional as purified.

Mutagenesis. Mutagenesis of UCH-L1 was performed using a combination of M13-based (Kunkel, 1985), cassette subcloning, and PCRTM methods. In M13 mutagenesis, UCH-L1 was excised from pRSET with XbaI and HinDIII (New England Biolabs, Beverly, MA) and inserted into M13mpl8 at the same sites.

Annealing, T7 polymerase extension (T7 Sequenase, USB), and ligation of primers (containing a new, silent HpaI site 5'to the mutation) with purified uracil-containing single-stranded M13 DNA generated the UCH-L1 H161D and H161Y mutants.

These mutants were identified by screening plaque minipreps (Sambrook et al., 1986) for susceptibility to HpaI digestion. The new HpaI site was then used to create the mutations H161Q, H161N, and H161K : degenerate cassettes produced by PCRTM were inserted by their HpaI and KpnI sites into pRSL 1 and sequenced. Lastly, C90S and D176N mutant PCRTM cassettes were made and inserted into the BssHII and DraIII (C90S) or BssHII and BsmI sites (D176N). In all cases, the cassettes were always smaller than 400 base pairs and were sequenced after insertion into the expression vector to verify the absence of Taq poly-merase-induced mutations. All isozymes and mutants were expressed in BL21 (DE3) cells, and the supernatants from lysozyme lysis were assayed. In most cases, the mutants were purified as above and their catalytic velocities and Michaelis constants were determined (Wilkinson et al., 1986).

UV-Vis, CD, and Raman Spectroscopy. UV-visible spectra from 190 to 800 nm were acquired on a CARY 219 dual-beam spectrophotometer. CD spectra were obtained on an Aviv Associates 62DS, using 10 or 1 mm path length quartz cuvettes (Hellma, Forest Hills, NY) at 25. 0 0. 1°C. Each spectrum was the average of five scan repetitions. CD spectra of the native protein were collected at 0. 95 mg of protein/mL (40 u. M) with 1 or 10 mm path length cells. To monitor ubiquitin binding by CD spectroscopy, ubiquitin and UCH-L3 (1 mL, 4 aM) were placed in separate compartments of a dual-compartment 9 mm cell, and the spectrum was recorded. The contents of the compartments were then mixed, and the spectrum was again recorded.

The former spectrum was subtracted from the latter to give the difference binding spectrum. A similar procedure was used for determining the effects of ubiquitin binding on the UV absorbance spectra, but with UCH-L3 and ubiquitin at 20 uM.

Circular dichroic spectroscopy was used to monitor the thermal denaturation of UCH at protein concentrations of 0. 1 g/L (4 uM) in 1 mm path length cells, or at 0.

1 g/L in 10 mm path length cells. The latter conditions were used for the H 161 K and H161Y mutants, which aggregated at higher concentrations. The temperature was controlled with a Hewlett Packard 89100A temperature controller equipped with an immersible temperature probe. The temperature scan rate was varied over a 4-fold range to confirm that measure-merits were made at equilibrium. Scans in both directions (heating and cooling) confirmed that the transitions measured were reversible. The fraction of native protein present at each temperature was calculated assuming a two-state transition between the initial and final spectra obtained, i. e., at any temperature the fraction of native species = (final ellipticity-observed ellipticity)/ (final ellipticity-initial ellipticity). Thermodynamic parameters were calculated from plots of In K. q vs 1/T or by curve fitting in Sigma Plot 4. 16 for Macintosh. Equilibrium constants were used to calculate thermodynamic state functions according to Kq = min + ( (max-min)/ (l + l/exp (s-h/x))) where x = T, s = aH/8. 314 J/K mol and h = AH/8. 314 J/mol.

Nonresonance Raman spectra were recorded using the 488 nm emission line of an argon laser (Spectra Physics model 165). Light scattered from the sample at 90° to the incident laser beam was dispersed by a holographic diffraction grating in a 0. 6 m triple monochromator (Triplemate, Spex Industries, Metuchen, NJ) and detected by an intensified photo-diode array detector (Princeton Instruments, Trenton, NJ). Power at the sample was less than 100 mW. The known Raman lines of toluene calibrated the system for each measurement, making the measured frequencies accurate to 1 cni 1.

Equilibrium Gel Filtration. Equilibrium gel filtration measurements were performed as described (Hummel and Dreyer, 1962) with the following modifications.

Tandem Superose 6 and 12 columns (0. 5 x 30 cm, Pharmacia) were equilibrated with running buffer (30 mM Tris-Cl, pH 7. 5 ; 5 mM DTT) containing 50 Fg of ubiquitin/mL. After equilibration with three column volumes, the ubiquitin concentration in the effluent was identical to that in the applied buffer. Purified UCH- L3 (100 uL, 5. 8 uM) was supplemented with ubiquitin to a final concentration of 50, ug/mL (5. 8 uM) and applied to this column. The concentration of ligand (ubiquitin) in the effluent was determined in triplicate by HPLC using a Waters WISP 710 B autoinjector and a Gilson HPLC equipped with a Spectra Physics SP4290 integrator (Wilkinson et al., 1986). To determine the effect of salt on ubiquitin binding, the studies were repeated in the presence of 0. 5 M NaCl.

RESULTS AND DISCUSSION UCH Isozyme Family. Ubiquitin C-terminal hydrolases comprise a small, newly defined, and novel family of thiol proteases. Among these, UCH-L3 is the best-characterized member. Human (Wilkinson et al., 1989) Drosophila (Zhang et al., 1993), and yeast (Liu et al., 1989) homologues have been described. These known UCH sequences are aligned in FIG. 14, where only residues found in at least three sequences are highlighted. All of these enzymes have slightly acidic isoelectric points (pI-5. 0) and molecular weights between 24 and 27 kDa. The numbering

system used here corresponds to the human UCH-L 1 residues. A number of areas in the sequence show a high degree of identity, most notably at positions 88-102 (the amino acid numbering system refers to the UCH-L1 sequence) (containing a conserved cysteine), 109-118, and 161-178 (containing a conserved histidine and an ELDGR sequence. Many of the positions in the aligned sequences are identical in all four sequences (44/249) or are similar in all four (52/249).

This degree of similarity in primary sequence and physical properties is usually taken as evidence of similar secondary and tertiary structure. In support of this assumption, all four UCH sequences give essentially identical plots of Kyte- Doolittle hydropathy. This suggests that the structural properties of these isozymes may be similar. The high homology also implies that the differential enzymatic specificity of each is a consequence of a few sequence differences at the substrate recognition site. A basal collection of UCH residues is probably necessary for proper folding and ubiquitin binding. These binding residues are expected to be on the surface of the protein and in regions that show significant sequence homology in the alignments shown in FIG. 14. Additionally, catalytic residues are expected to be near the surface but are generally at the bottom of a cleft or invagination of the protein surface. To examine these relationships and make predictions about which residues to mutate, the secondary structure for this protein family has been predicted by submitting the aligned sequences shown in FIG. 14 to the PredictProtein server (PredictProtein@EMBL-Heidelberg, DE). This method of prediction uses a neural net and preserves the information content of the aligned sequences, as well as that of surrounding residues rather than using only a single consensus residue at each position (Rost and Sander, 1993). These predictions (with an 82% level of confidence) are given in the last row of FIG. 14 and are consistent with analyses by the Raman and circular dichroic spectroscopies discussed below.

Interestingly, the putative active site cysteine at position 90 in UCH-L1 (see below) is flanked by two putative hydrophobic (3-sheet regions. These two regions of

p-sheet may span from the surface of the molecule to a more protected site deeper in the molecule and position the active site thiol in the expected catalytic cleft. The cysteine is juxtaposed between the very small residues, alaine and glycine. They may allow the approach of a scissile peptide bond to form the tetrahedral intermediate. If the inventors presume the mechanism of this protease to be papain-like. then there must also be a conserved histidine which can act as a catalytic base, polarizing the sulfhydryl and enhancing its nucleophilicity. Two positions in the UCH family have conserved histidines, these being positions 97 and 161 in UCH-L I. H97 is unlikely to be involved since it is only seven residues removed from the active site and at the opposite end of the predicted P-sheet. In contrast to papain and the serine proteases, thiol proteases of the interleukin-converting enzyme (ICE) family do not position a third residue to hydrogen bond to the catalytic histidine (Walker et al., 1994). Thus, it is not known if a"catalytic triad"is involved in catalysis by the UCH gene family.

UCH Expression and Purification. Recombinant proteins were expressed in E. coli using a modified pRSET vector (Invitrogen). The modification removed the coding region for the oligohistidine leader sequence present in the parent vector.

Expression in this system is driven by a T7 RNA polymerase promoter, with induction of the polymerase by IPFG. Upon induction, the UCH isozymes and mutants were expressed at 15%-30% of the soluble protein. The enzymes were expressed, purified (see Experimental Procedures), and assayed for kinetic parameters.

To identify the active site residues involved in UCH catalysis, the inventors have mutagenized the wild type UCH-L 1 cDNA. The vector encoding this UCH isozyme is more tractable for mutagenesis (compared to UCH-L3) because of its greater number of useful restriction sites. Several mutants were made whose properties are summarized in Table 5. In every case, UCH-L 1 mutant proteins were produced in amounts equal to the wild type enzyme, based on SDS-PAGE analysis of expression lysates. The inventors assayed each expression lysate for activity, and active mutants were purified as described.

TABLE 5 : Mutagenesis and Kinetics of UCH-L1 Mutants Relative Rate Mutant Codon Change (Velocity/wt Velocity) K, ( ; iM) wild type 1. 00 1. 20 C90S TGT-). TCT <lxl0' nd H97Q CAC CAA 0. 85 0. 65 H97N CAC # AAC 0. 87 0. 60 HI61D CAT GAC 8. 5 x 10-5 1. 50 H161K CAT # AAA <1#10-7 nd H161N CAT # AAC <1 # 10-7 nd H161Q CAT # CAA <1 # 10-7 nd H161Y CAT # TAC <1 # 10-7 nd D176N GAT # AAT 0. 025 7. 40 Q73R CAA # CGA 0. 97 1. 10 Active mutants were purified as described for the wild type enzyme (see Experimental Procedures). Hydrolysis rates are the average of two determinations at 15 pM UbOEt, or were Michaelis constants determined according to Wilkinson et al. (1986). Wild type UCH-L1 velocity is 25 pmol/min/mg vs ubiquitin ethyl ester (nd : not determined).

Identification of the Active Site Cysteine. The inventors examined the effect of changing the putative active site thiol (C90) to a serine. This cysteine residue is conserved among all UCH's and was suspected to be involved in catalysis, though direct proof of this residues role in catalysis has not yet been shown. The inventors generated a UCH-L1-C90S mutant (see Procedures). Assay of the bacterial lysate expressing UCH-L1-C90S showed no detectable activity. To quantitatively assess the upper limit of this activity, the C905 mutant was purified and assayed. Even at

equimolar enzyme to substrate ratio (17 uM), the half-life of the substrate is over 4. 5 h. Because serine is isoelectronic with cysteine, it is likely that this abrogation of activity is a direct effect and not the result of a structural change. In support of this, the C905 mutant exhibits a thermal denaturation profile with thermodynamic parameters nearly identical to the native enzyme (see below). Therefore, cysteine 90 is directly involved in catalysis, probably as the active site nucleophile.

Identification of an Active Site Histidine. The inventors next sought to identify the active site histidine. Two positions in the alignment have a conserved histidine, corresponding to H97 and H161 in UCH-L1. To determine if these were important to catalytic function, the inventors conservatively mutated H97 to a glutamine or asparagine. These carboxamide residues cannot provide a general base for catalytic function, but could provide hydrogen bonding similar to the NI or N3 imidazole nitrogens and hence could provide a structural replacement. Purified UCH- Ll H97Q and H97N catalytic velocities are approximately 85% as rapid as the wild type enzyme (Table 5 and Experimental Procedures). This suggests that H97 is not involved in catalysis.

The inventors then mutated the other fully conserved histidine at position 161.

In short, all H161 mutants were either catalytically inactive or very significantly impaired. H161Q, H161N, H161Y, and H161K possess no measurable esterase activity down to the detection limit of the inventors'assay. These mutants are minimally seven orders of magnitude slower than the wild type hydrolase. Individual H 161 mutations could be expected to supply an adequate structural replacement for positive electrostatic charge (lysine), hydrogen bonding by the imidazole ? t (asparagine) andr (glutamine) nitrogens (Vaaler and Snell, 1989), or aromaticity and steric volume (tyrosine). Interestingly, UCH-L1 H161D shows detectable activity, about 4 orders of magnitude less than that of native enzyme. Determination of the Km of this purified mutant showed that only the reaction rate was altered and that the Km was unchanged (Table 5). In this context a carboxylate may function as a general

base or a hydrogen bond acceptor. Either interaction would abstract proton density from the nearby cysteine thiol and enhance its nucleophilicity. Since neither H161N nor H161Q can support this level of catalysis, but could hydrogen bond, the inventors favor a direct role for D 161 as a general base. This would be the first example of a functional cys-asp dyad in a protease, though the velocity of catalysis is small.

UCH-Ll Hl61D shows CD spectra typical of native UCH-L1 (described below), suggesting that this residue is not important for the gross enzyme structure. The inventors'data therefore indicate that histidine 161 is intimately involved in catalysis, probably as an active site general base.

Mutation of the ELDGR Box. Because the binding of ubiquitin to UCH is primarily electrostatic (shown below), and since acid residues may be involved in catalysis, the inventors mutated a universally conserved aspartate in the most conserved area of the UCH sequence, the ELDGR box. D176 was changed to an asparagine, resulting in a sterically unaltered charge mutant in a highly conserved region. This mutant shows a significant, measurable activity of 2. 5% wild type. To determine if the drop in catalytic rate was due to an effect on binding strength, the Km was determined (Wilkinson et al., 1986). Progress curve kinetics (Wilkinson et al., 1986 ; Orsi and Tipton, 1979) show this mutant to have a Km = 7. 4 uM, approximately 6-fold weaker than that of the native enzyme. The inventors find that the calculated specificity constant Vit,, is 250-fold lower than the wild type Ll. Catalytic efficiency is thus lowered dramatically, but is not obliterated, and this might be expected for a residue not directly involved in catalysis. The ELDGR box may therefore be involved in the formation of a binding site or the orientation of the substrate.

Mutation of Q73. The amplification of the UCH-L1 coding sequence by RT- PCRTM resulted in two errors. One of these changes, a G to C transversion affecting V200, was silent and was not repaired. Another G to A transition generated the mutant Q73R. The inventors repaired the R73 mutation by replacing the defective

region with a fragment from the rat UCHL1CDNA. Both rat and human proteins have identical sequences in this region (Kajimoto et al., 1992 ; Day et al., 1990), and the swap thus repaired the original PCRTM mutation (Experimental Procedures).

Residue 73 is 17 residues N-terminal to the active site cysteine. It is predicted to be at the surface of the enzyme, possibly as part of a turn at the opposite end of the (3-sheet anchoring the active site cysteine. Since all known UCH sequences have a Q in this region (equivalent to either position 73 or 74 in UCHL1), it was of interest to examine the catalytic activity of this mutant. Table 1 shows that mutation of this position to the positively charged R residue had no effect on the activity of the enzyme or its affinity for substrate.

Structural Effects of These Mutations. To ensure that the lack of activity in these mutants was not due to gross structural misfolding of the enzymes, the inventors analyzed selected mutants by circular dichroism. UCHL1 mutants Q73R, H161D, D176N, and C90S all show CD spectra typical of UCH-L1 (described below), suggesting that these residues are not important for the gross enzyme structure. All of these mutants were expressed at levels similar to the wild type, again suggesting that folding and solubility were not problems with these specific mutations.

Binding of Ubiquitin to UCH-L3. To characterize the ubiquitin binding site and to identify any structural changes and/or perturbation of the environment of amino acid side chains associated with the binding of ubiquitin to UCH, the inventors have studied the spectral properties of the more soluble isozyme, UCH-L3, upon binding of ubiquitin. Circular dichroism has previously been used to monitor protein-protein interactions accompanied by conformational changes, as well as to examine the environment of aromatic residues (Beltramini et al., 1992 ; Blazy et al., 1992 ; Grobler et al., 1994 ; Vuillemier et al., 1993). The inventors purified UCH-L3 (Experimental Procedures) and used it to study substrate binding by various approaches. The inventors were unable to detect any changes of ellipticity in CD difference spectra upon binding of ubiquitin and UCH-L3. This suggests that the structure of the two

proteins are not altered by binding, such that no gross"induced fit"conformational changes are detectable.

To examine if aromatic residues were perturbed by substrate binding, the W spectra of Ub and UCH-L3 were recorded in dual-compartment cells. After the compartment contents were mixed to initiate binding, no significant spectral change was seen relative to the unmixed control. The data from UV and CD spectra cannot distinguish minor tertiary structure alterations in UCH, and the inventors cannot comment on this possibility solely on their basis. The data do suggest that the electronic environments of the aromatic side chains are not radically altered by ubiquitin binding. Above 340 nm, the lack of UV absorption is consistent with the absence of chromophoric prosthetic groups in the enzyme. The spectra of UCH-L3 yield a Beer-Lambert extinction coefficient of 21 000 L/tool cm at 280 nm. UCH-L1 exhibits similar spectral characteristics, with an extinction of 15 600 L/tool cm. These data are consistent with the expected extinction based on the aromatic content of the polypeptides.

Binding of ubiquitin to UCH-L3 was not detectable by any of the spectral methods used above. Nonetheless, kinetic evidence predicts a sub-micromolar binding constant (Wilkinson et al., 1986). Additionally, it is known that the enzyme is specifically bound to and eluted from a ubiquitin affinity column (Duerksen-Hughes et al., 1989 ; Pickart and Rose, 1985). The kinetically obtained Km must not be interpreted as a substrate dissociation constant, and the ubiquitin affinity column cannot be used to quantify the binding strength. Thus a direct gel filtration approach was used to monitor this binding. In these studies, the column buffer is equilibrated with ligand ubiquitin and the enzyme sample is supplemented with an equal concentration of ligand. If binding occurs, one expects to observe a peak of ligand at the elution position of the enzyme and a depressed level of ligand at the included volume of the column. FIG. 15A shows that purified UCH-L3 is 91% occupied by ubiquitin when chromatographed in the presence of 5 ILM

ubiquitin-containing buffer. Integration of the peak area shows that 3. 45 nmol of ubiquitin was bound to the 3. 80 nmol of UCH-L3 applied. An apparent binding constant of 0. 5 uM can be calculated from these data. This is similar to the Km for UbOEt (Wilkinson et al., 1986) and implies that most of the binding energy is due to ubiquitin alone and not the ester functionality. These data demonstrate that UCH-L3 possesses only one binding site with a micromolar Kd and that the stoichiometry of binding is 1 : 1.

The above data demonstrate the binding of ubiquitin to UGH-L3 and suggest that there are few gross structural changes associated with this binding. Further, the environment of aromatic residues is not greatly perturbed. This suggests that polar interactions may be important for the binding. Indeed, the inventors have noted that increased ionic strength inhibits hydrolysis of ubiquitin ethyl ester. This inhibition is virtually complete at 10 uM substrate and 0. 40 M NaCl. To examine if the inhibition by ionic strength was due to decreased substrate binding, or to a change in the catalytic properties of the protein, the inventors have repeated these binding studies in the presence of inhibitory levels of salt. Ubiquitin binding is completely abrogated in the presence of 0. 5 M NaCl (FIG. 15B). The structure of the enzyme is not grossly perturbed by the presence of salt, as the CD spectra of UCH-L3 in 0 and 0. 5 M NaCl are virtually identical. These data suggest that the binding interactions of the enzyme and substrate are primarily electrostatic and not hydrophobic.

Spectroscopic : Analysis of UCH-L3. Circular dichroism spectroscopy was used to estimate the amount of secondary structure motifs in UCH isozymes and mutants (FIG. 16) and to evaluate the effects of mutation on the folded protein structure. The CD spectra show an absolute minima at 222 nm, characteristic of the presence of a-helices. This is also confirmed by the relative minima at 208 nm and absolute maxima at 202 nm (Johnson, 1988). Calculating the mean residue ellipticity, at 208 and 222 nm, the inventors obtain values of 12 090 and 9160 deg cm2/dmol, respectively. Using the sum of structures constraint (Greenfield and Fasman, 1969)

these values predict a-helix contents of 31. 2% and 32. 4%. Also shown in FIG. 16 is the near-UV dichroism due to the chiral environment of the aromatic residues (curve labeled x 100). As is typical, this region shows much less ellipticity (-60 deg cm2/dmol), but since this region might serve as an environmentally sensitive reporter for the aromatic residues the inventors have shown it.

Finally, classical nonresonance laser Raman spectroscopy was also used as a structural probe. FIG. 16 shows the Raman spectra of UCH-L3 from 400 to 1750 cm' 1. The inventors used two methods to calculate the amounts of structural motifs which are based on the conformationally sensitive nature of the peptide carbonyl stretch absorbance. The spectral bandwidth, intensity, and position of this amide I Stokes emission were used to estimate quantities of four generic secondary structures : helix, P-sheet, turn, and random (Alix et al., 1981). This method suggests 48% helical content, 25% R-sheet, 16% turn, and 11%"other". Another method (Lippert et al., 1976) uses the spectral characteristics (1240, 1632, and 1660 cm''transitions) of pure helix, (3-sheet, and random forms of poly L-lysine to calculate the secondary structure content. The inventors'data predict 40% helix, 43% (3-sheet, and 17% random coil when analyzed in this way, but this method cannot distinguish between (3-turn and R-sheet motifs. These predictions therefore concur generally with predictions based on Alix et al. (1988) and also with the CD data presented above. Weighting the Raman, CD, and prediction algorithms equally, approximate averages of 38% helix, 22% (3-sheet, 18% turn or loop, and 19%"nonordered"secondary structures are obtained. Minor discrepancies between the methods may arise as a consequence of the"sum of structure"constraints or from the nature of the model compounds used as the basis for the various computations described above.

The inventors'data show that UCH isozymes possess both helix and R-sheet motifs, similar to the papain family of thiol proteases. To date, the solution crystal structures of five thiol proteases have been solved. Three of these, papain, calotropin Dl, and actinidin, are from plant sources ; two others, liver cathepsin B and the

interleukin 1- (3-converting enzyme"ICE", are from mammalian sources [reviewed by Walker et al. (1994). These enzymes differ from the all-p-chymotrypsin class of serine proteases in both catalytic residues and overall structure. ICE and subtilisin both possess helical content, however, and exhibit an antiparallel p-sheet core domain.

While UCH enzymes resemble the papain family members in size and secondary structure content, sequence comparison with the papain family suggests that the UCH family should be classified as a distinct gene family. The solution of a UCH crystal structure would provide a valuable addition to the small collection of a/p-proteases, and these studies are ongoing.

Thermal Denaturation. The above results demonstrate that the recombinant enzymes and mutants display normal spectroscopic properties at room temperature.

This suggests that all mutants tested fold correctly and are soluble under these conditions. However, the temperature of the enzymatic assay and normal physiological environment of these enzymes is 37°C. To demonstrate that the loss of activity was due to a direct effect and not irreversible unfolding of the enzymes at assay temperature, the inventors have conducted thermal denaturation studies monitoring the 222 nm circular dichroism signal. Using this technique, the thermodynamics of protein denaturation have been studied for several enzymes Alexander et al., 1992 ; reviewed by Privalov and Gill (1988). This method provides a powerful, general tool for assessing the structural stability of enzymes and mutants. FIG. 17 shows the temperature-dependent changes in the 222 nm CD signal of UCH-L1. As can be seen, there is a thermal transition at approximately 52°C resulting in a 45% diminishment in this conformationally sensitive signal. UCH-L3 is also subject to the same transition, though the loss of ellipticity is slightly less. Cooling the sample results in the restoration of the original spectra, and wavelength scans at 65°C are typical of proteins with high random coil content. Also, the transition is fully reversible if the protein concentration is less than 100 , g/mL (10 gghnl for H161K and H161Y) and if the protein is not allowed to remain denatured for more than 5 min before the temperature is lowered.

These data can be analyzed according to a two-state model, and the relevant thermodynamic parameters can be calculated. The inset to FIG. 17 shows the Arrhenius plot of the data. As obtained from the replot, this transition is characterized by values of AH= 1. 56 kJ/mol of residue, AS = 4. 80 J/K mol of residue, and AG 28. 6 kJ/mol of UCH-Ll at 25°C. It is assumed that this transition is the reversible denaturation of UCH. The rather modest stability, of this protein is consistent with the reversible folding of a single domain protein. Many small globular proteins exhibit folded states stabilized by only 20-60 kJ/mol of Gibbs free energy (Privalov, 1979).

TABLE 6 Thermodynamics of Denaturation of UCH's" Melting Point Enthalpy, AH Entropy, AS Gibbs Energy, AG Enzyme (Tm 0. 2°C) (kJ/mol of aa) (J/K mol of aa) CJ/mol of UCH) UCH-L3 50. 9 1. 15 3. 52 21. 7 UCH-L1 51. 8 1. 56 4. 80 28. 6 UCH-L1 C90S 51. 5 1. 55 4. 78 27. 7 UCH-Ll Hl61D 49. 9 1. 50 4. 69 22. 6 UCH-L1 H161K 52. 7 1. 07 3. 30 19. 1 UCH-Ll H161Y 52. 7 1. 10 3. 40 19. 2 'Melting points are derived from the primary denaturation data.

Thermodynamic values for denaturation are calculated as described in the text, where AH = kJ/mol of amino acid residue, AS = J/K mol of amino acid residue, and AG = kJ/mol of UCH at 25°. Conventions are according to Privalov (1979).

The inventors also performed this thermodynamic analysis for the UGH-L3 isozyme and the Ll isozyme mutants C90S, H161D, H161K, and H161Y. In general the wild-type and mutant enzymes have virtually indistinguishable circular dichroism spectra and only slightly differing denaturation curves. All denature at 50-53°C,

where the melting point is defined as that point in the thermal denaturation curve where Keq = 1, i. e., the midpoint. Thermodynamic values thus derived are shown in Table 6. By comparison, the neuron specific UCH-L1 appears slightly more stable than its hemopoeitic homologue, UCH-L3. Wild type LI and the isoelectronic mutant C90S both show virtually identical melting points and thermodynamic stabilities per residue (Privalov and Gill, 1988) with AG : 28. 6 and 27. 7 kJ/mol at 25°C, respectively.

Mutations at the catalytic histidine were only slightly destabilizing. as determined by a melting point depression (H161D) or unfavorably altered thermodynamic state functions (H161K and H161Y). On the basis of these data, the inventors conclude that the inactivity of the C90 and H161 mutants is due to the loss of important catalytic residues and not due to misfolding or a decreased stability of the folded form.

Given the above observations, the present invention also contemplates constructing columns containing a matrix material that has immobilized enzyme bound to its surface. Such a column may be constructed such that it is much more likely than not that the immobilized enzyme, whether UCH-L1, UCH-L3, or variants thereof, shows greater catalytic activity as opposed to unbound enzyme. Also contemplated is protecting the face of the enzyme utilizing a cross-linked ubiquitin- protein or ubiquitin-peptide complex. The binding of such a complex to the enzyme is generally tight and specific, and if cross linked, the complex serves as a protector of the active site. To activate the enzyme, one may disassociate the ubiquitin with, for example, a high salt concentration. Such columns will find use in catalytically cleaving and then separating ubiquitin from the peptides and small proteins that are part of the fusion protein. Conversely, if one wanted to further purify the enzyme, one could employ a column having ubiquitin bound to its matrix, and more preferably, ubiquitin that has been cross linked.

The inventors have presented data to demonstrate that cysteine 90 and histidine 161 are the active site nucleophile and general base involved in UCH-L1

catalysis. These data assist in crystalographic model building, as the two residues must be juxtaposed in the tertiary structure and will define the active site. It can also be safely assumed that the other isozymes of the UCH family possess the same catalytic chemistry and residues, for reasons described above. The electronic nature of the binding suggests that one of two faces of ubiquitin is involved in an extensive interaction with this enzyme. One face has been defined as an"acidic face"with many such clustered on the surface of the a-helix from residues 20 to 34. Many of the basic residues are clustered on the opposite face of the molecule. It is not immediately obvious which face is contacting the surface of the enzyme, although there are several approaches which could be pursued to further define this. It is interesting to note that the majority of amino acid substitutions across species occur in the"acidic"face of ubiquitin (Wostmann et al., 1992), and for this reason, the inventors assume that the"basic"face is involved in these binding interactions. Data from Burch and Haas (1994) suggest that R42 of ubiquitin is involved in recognition by UCH-L3. Also, the aspartate in the conserved ELDGR box may be involved in the binding. The effect of the D176N mutation on the Michaelis constant for ubiquitin shows that this residue may participate in an ionic interaction with ubiquitin or provide minor"orienting"effects for the fine tuning of substrate positioning. Rose and Warms (1983) have also shown that the two C-terminal glycine residues are necessary for effective inhibition of UCH-L3 by ubiquitin. The inventors find that the attachment of a hexahistidine motif to the N-terminus of ubiquitin does not affect hydrolysis rates to any measurable extent.

In summary, the inventors'data suggest that UCH isozymes (a) utilize cysteine 90 as the nucleophile, (b) use histidine 161 as the general base catalyst, (c) bind ubiquitin electrostatically, (d) bind the intact ubiquitin C-terminus, (e) may possess a carboxylate P3 binding pocket for arginine, (f) do not bind the amino terminus of ubiquitin, (g) bind other basic residues in ubiquitin, and (h) utilize several of UCH's acidic residues in binding. These studies are useful in building models of the enzyme for crystallographic and structural studies, for defining the enzyme-substrate

interaction, and in site-directed mutagenesis studies designed to alter recognition and specificity of these enzymes.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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