Crystal Structure of an Inhibitor Complex of the 3C Proteinase from Hepatitis a Virus (HAV) and Implications for the Polyprotein Processing in HAV

Virology 265, 153–163 (1999) Article ID viro.1999.9968, available online at http://www.idealibrary.com on

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provided by Elsevier - Publisher Connector Crystal Structure of an Inhibitor Complex of the 3C Proteinase from Hepatitis A Virus (HAV) and Implications for the Polyprotein Processing in HAV

Ernst M. Bergmann,*,† Maia M. Cherney,*,† John Mckendrick,‡ Sven Frormann,‡ Colin Luo,* Bruce A. Malcolm,*,1 John C. Vederas,‡ and Michael N. G. James*,†,2

*Department of Biochemistry, †Medical Research Council of Canada Group in Protein Structure Function, and ‡Department of Chemistry, University of Alberta, Edmonton, Alberta Canada, T6G 2H7 Received May 7, 1999; returned to author for revision June 14, 1999; accepted August 11, 1999

The proteolytic processing of the viral polyprotein is an essential step during the life cycle of hepatitis A virus (HAV), as it is in all positive-sense, single-stranded RNA viruses of animals. In HAV the 3C proteinase is the only proteolytic activity involved in the polyprotein processing. The specific recognition of the cleavage sites by the 3C proteinase depends on the amino acid sequence of the cleavage site. The structure of the complex of the HAV 3C proteinase and a dipeptide inhibitor has been determined by X-ray crystallography. The double-mutant of HAV 3C (C24S, F82A) was inhibited with the specific inhibitor iodoacetyl-valyl-phenylalanyl-amide. The resulting complex had an acetyl-Val-Phe-amide group covalently attached to the S␥ atom of the nucleophilic Cys 172 of the enzyme. Crystals of the complex of HAV 3C (C24S, F82A) acetyl-Val-Phe-

amide were found to be monoclinic, space group P21, having 4 molecules in the asymmetric unit and diffracting to 1.9-Å resolution. The final refined structure consists of 4 molecules of HAV 3C (C24S,F82A) acetyl-Val-Phe-amide, 1 molecule of DMSO, 1 molecule of glycerol, and 514 water molecules. There are considerable conformational differences among the four molecules in the asymmetric unit. The final R-factor is 20.4% for all observed reflections between 15.0- and 1.9-Å resolution

and the corresponding Rfree is 29.8%. The dipeptide inhibitor is bound to the SЈ1 and SЈ2 specificity subsites of the proteinase.

The crystal structure reveals that the HAV 3C proteinase possesses a well-defined SЈ2 specificity pocket and suggests that

INTRODUCTION polyprotein (Graff et al., 1999; Harmon et al., 1992; Jia et al., 1993; Martin et al., 1995, 1999; Schultheiss et al., 1994, In all positive-sense, single-stranded RNA viruses that 1995a, 1995b; Stanway and Hyypia¨, 1999). infect animals, the specific processing of the viral The picornaviral 3C proteinases are related to the polyprotein is a central step of the viral life cycle (Kra¨us- chymotrypsin-like serine proteinases (Gorbalenya and slich and Wimmer, 1988). The viral polyprotein is gener- Snijder, 1996). However, the 3C proteinases are cysteine ated by direct translation of the viral RNA genome. In the proteinases; the nucleophile is the sulfur atom of a cys- viruses of the family Picornaviridae the polyprotein pro- teine residue within the conserved two-domain ␤–barrel cessing occurs cotranslationnally and is sequential (Pal- structure of the chymotrypsin-like enzymes (reviewed by menberg, 1990). Usually, the first cleavage during the Bergmann and James, 1999b; Gorbalenya and Snijder, sequential polyprotein processing separates the struc- 1996; Ryan and Flint, 1997). tural proteins from the other gene products (Ryan and Specific recognition of the cleavage sites within the Flint, 1997). In all picornaviruses, the 3C gene product viral polyprotein by the 3C proteinase depends on the performs the majority of the proteolytic cleavages that amino acid sequence of the cleavage site. Cleavage liberate the individual viral gene products. The details of sites within the picornaviral polyprotein are typically the polyprotein processing differ among the six known characterized by the sequence of the four residues pre- genera (Bergmann and James, 1999a). In the hepatovi- ceding the scissile peptide bond and the two residues ruses, and presumably also in the viruses of the new following it (Seipelt et al., 1999, and references therein). genus parechovirus, the 3C gene product is the only We use the nomenclature of Schechter and Berger (1967) proteolytic activity involved in polyprotein processing to describe the interactions of the peptide substrate and cleaves all the specific cleavage sites within the viral sequence with the specificity subsites in the active site ͉ Ј Ј of the proteinase. P4P3P2P1 P1P2 represents the residues of a specific substrate and S to SЈ represent the corre- 1 Current address: Schering-Plough Research Institute, 2015 Gallop- 4 2 ing Hill Road, Kenilworth, NJ 07033. sponding specific binding sites on the enzyme. 2 Ј To whom correspondence and reprint requests should be ad- Hexapeptide substrates (P4 to P2) bind to the active site dressed. Fax: (780) 492-0886. E-mail: [email protected]. of chymotrypsin-like proteinases in a canonical confor-

Ј Ј mation (Bode and Huber, 1992; Perona and Craig, 1995; other positions, including P1 and P2, contributes to the Read and James, 1986). As a result of this main chain determination of the sequence of cleavages. The only conformation the side chains of several residues point amino acid substitution, within the sequences of the into specificity pockets on the surface of the enzyme. The cleavage sites of the HAV polyprotein, that distinguish a picornaviral 3C proteinases possess well-defined S4,S2, faster-growing, cell culture-adapted HAV strain is a ly- Ј and S1 specificity pockets (Bergmann et al., 1997; Mat- sine to asparagine mutation in the P2 position of the thews et al., 1994; Mosimann et al., 1997). The side primary cleavage site of the viral polyprotein (Zhang et chains of the P5 and P3 residues point away from the al., 1995). Likely other factors that contribute to the de- active site into solvent. This correlates well with the fact termination of the sequence of cleavage events are the that the 3C cleavage sites within picornaviral polypro- accessibility and the conformation of the cleavage sites teins can be identified by the residues in the P4,P2, and in the folded polyprotein.

P1 positions (Seipelt et al., 1999). In addition, rhino- and Here we present the cocrystal structure of a covalent enteroviral 3C proteinases show preferences for a gly- complex of the HAV 3C proteinase with an inhibitor, de- Ј Ј Ј cine residue in the P1 position of a substrate. signed to bind specifically to the S1 and S2 sites of the Cocrystal structures of picornaviral 3C proteinases enzyme (Mckendrick et al., 1998). The preferred dipeptide with inhibitors specifically directed against the P4-to sequence was identified by incubating the enzyme with a

P1-specific subsites confirmed that these substrate res- mixture of inhibitors and analyzing the result by mass spec- idues are indeed bound in the canonical conformation troscopy. HAV 3C was able to select the tightest binding found in chymotrypsin-like proteinases (Dragovich et al., inhibitor from the mixture, thereby providing evidence for Ј Ј 1998a,b; Webber et al., 1998). Furthermore, prior to the specificity in the recognition of the P1 and P2 residues. elucidation of any cocrystal structure of a 3C proteinase Unfortunately, for technical reasons, it was not possible to inhibitor complex, models of substrate binding, based on include all amino acids in each position of the dipeptide this conformation, could successfully explain the speci- inhibitor in the mixtures used (Mckendrick et al., 1998). The ficities of 3C proteinases (Bergmann et al., 1997; Mat- best dipeptide sequences found from the inhibitors used Ј Ј thews et al., 1994; Mosimann et al., 1997). These models were the sequences P1-P2 Ser-Phe and Val-Phe. The crystal did not predict the specific interactions, if any, of the PЈ structure reported here is for the inhibitor acetyl-valyl-phe- residues of a substrate. In all the inhibitor cocrystal nyalanyl-amide covalently bound to the S␥ atom of the structures of 3C proteinases published so far, the spe- nucleophilic residue Cys172. The proteinase inhibitor com- cific interactions of the inhibitor and the enzyme are with plex derives from the reaction of the nucleophilic thiol of the the P5 to P1 sites or less. No small-molecule inhibitor proteinase with an iodoacetyldipeptide. The reaction was cocrystal structure for any chymotrypsin-like proteinase expected to be similar to the alkylation reaction of iodo- reveals binding to the PЈ sites and the conformation in acetamide with sulfhydryls in proteins. Confirmation of this which the PЈ residues bind to these proteinases can only mechanism of inhibition had been obtained by mass spec- be inferred from cocrystal structures with large protein trometry prior to the elucidation of the crystal structure inhibitors (Bode and Huber, 1992; Read and James, 1986). (Mckendrick et al., 1998). Typical cleavage sites of the 3C proteinases from Based on the details of the specific interactions be- hepatitis A virus (HAV) are distinguished by a consensus tween this dipeptide inhibitor and the specific subsites of L S ͉ sequence /IX /TQ XX (reviewed by Bergmann, 1998, and the HAV 3C proteinase, we propose a mechanism that Seipelt et al., 1999). The most important determinant is contributes to the preferential recognition of the primary the glutamine residue in the P1 position. Two of the cleavage site within the HAV polyprotein. proposed cleavage sites within the HAV polyprotein have ͉ aP1 glutamate residue. These two cleavage sites (VP1 X RESULTS AND DISCUSSION and 3A͉3B) have not been experimentally confirmed and Crystallization, structure solution, and refinement these recognition sequences constitute poorer substrates for the 3C proteinase (Jecht et al., 1998; Probst et Crystals of the HAV 3C acetyl-Val-Phe complex were al., 1998). Recently, experimental evidence has been pre- found to belong to the monoclinic space group P21 and sented, demonstrating that VP1͉X is not cleaved by the to diffract to a maximum resolution of 1.9 Å. The statistics 3C proteinase in HAV (Graff et al., 1999; Martin et al., of the diffraction data are shown in Table 1 and the 1999). It is also clear that the primary cleavage site of the results of the refinement are summarized in Table 2. HAV polyprotein, which separates the structural and non- Initial electron density maps revealed a covalently bound structural proteins, is located at the N-terminus of the 2B molecule of the inhibitor in the active site of each of the gene product and has the sequence GLFSQ͉AKI (Jia et four molecules, as had been expected from mass spec- al., 1993; Martin et al., 1995; Schultheiss et al., 1994, trospcopy of the inhibited enzyme (Mckendrick et al., 1995b). What determines the sequence of cleavages in 1998). The dipeptide inhibitors are bound in the same

HAV among the seven sites with glutamine in the P1 location in each of the four independent molecules (Fig. position is not clear. Possibly the type of residue in the 1a). Fifteen cycles of manual rebuilding and computa- INHIBITOR COMPLEX OF 3C PROTEINASE FROM HEPATITIS A VIRUS 155

TABLE 1 TABLE 2 Crystal Parameters and Statistics of Diffraction Data Refinement and Model Statistics

Space group P21 (4 molecules/asymmetric unit) No. of reflections 59,384 (͉Fo͉ Ͼ 0.0␴ and 15.0- to 1.9-Å Unit cell a ϭ 50.56 Å; b ϭ 78.35 Å; c ϭ 105.29 Å resolution) ␣ ϭ 90°; ␤ ϭ 97.5; ␥ ϭ 90° No. of nonhydrogen atoms 7,228 (including 514 water molecules) Resolution 15.0–1.9 Å (1.96–1.9 Å)a No. of degrees of freedom 28,832 Observations 494,113 R-factora 0.204 Unique reflections 59,384 (4001) Free R-factorb 0.298 Completeness 92.3% (74.7%) Root-mean-square deviations Average intensity I/␴(I) 4.8 (1.8) from ideal geometry b Rmerge 0.033 (0.157) Bonds 0.012 Å Bond angles 1.456° a Numbers in parentheses are for highest resolution shell. General planes 0.025 Å b ϭ ¥ ¥ ͉ Ϫ͉͗͘ ¥ Rmerge hkl ( i Ii I / iIi). No. of ␸, ␺ angle outliers 49 (6.2%)

a R ϭ ¥hkl (͉͉Fo͉ Ϫ ͉Fc͉͉)/¥hkl͉Fo͉ for 55,982 reflections used in the refinement. tional refinement resulted in a final model consisting of b For the 3902 reflections not used in the refinement (6.5%). residues A1 to A213, B1 to B217, C1 to C213, D1 to D217, 4 inhibitor molecules bound in the active sites of all 4 molecules, 1 molecule of DMSO, 1 molecule of glycerol, Differences between the four molecules in the and 514 ordered water molecules. Residues 214–217 in asymmetric unit molecules A and C are presumably disordered and not visible in the electron density maps. The results of the There are considerable differences among the four refinement are summarized in Table 2. Representative molecules in the asymmetric unit (Fig. 2). There are also electron density for the inhibitor bound in the active sites differences in the conformations of the bound inhibitors of molecules D and B is presented in Figs. 1b and 1c. (see below). The most prominent differences among the Tests during the refinement of the structure, in particular four independent molecules manifest in the turns and the behavior of the free R-factor, indicated that restraints loops that connect the ␤-strands of the two domains and for the noncrystallographic symmetry should not be ap- are involved in the intermolecular interactions that form plied. the crystal. Residues 20–23, 48–53, 77–83, and 140–160 The enzyme used in the present study is a double- and the C-terminus residues 214–217 constitute the re- mutant, C24S, F82A, that had been designed based on gions with the most significant conformational differ- the structure of the native enzyme. In the crystal structure ences (Fig. 2). of native HAV 3C Phe 82 contributed to an intermolecular Residues 77 to 83 form the loop connecting ␤-strands interaction in one of the two unique molecules of the eI2 and fI. The conformation of these residues varies dimer. In the other molecule within the asymmetric unit among all the molecules of HAV 3C in the crystal struc- dimer the loop with Phe 82 was flexible and did not tures of HAV 3C. This is true for the structure presented participate in any intermolecular interactions (Bergmann here, as well as for the native structure (Bergmann et al., et al., 1997). The enzyme for the crystal structure pre- 1997). What determines the conformation of this loop is sented here is a Phe82Ala mutation that was designed to the environment of the HAV 3C molecule and the inter- change the crystal interaction and produce a different molecular interactions it forms. In the crystal, these are crystal form. The crystal form and the packing environ- the intermolecular interactions that form the crystal ment within the crystal in the present study are indeed packing; in a cellular environment, this would likely be different. other proteins with which HAV 3C interacts. This could

FIG. 1. Binding of the acetyl-Val-Phe inhibitor in the active site of HAV 3C. (a) Ribbon representation of the secondary structure of molecule D of the structure of HAV 3C (C24S, F82A) acetyl-Val-Phe-amide in stereo. The bound inhibitor is included in a bond-stick representation and colored in yellow. The ␤-strands of the N-terminal domain are colored lilac and those of the C-terminal domain in cyan. The ␤-strands of the anti-parallel ␤-ribbon that extends from the C-terminal domain are colored in light gray. Helices are colored in green and coil structures in red. The residues of the oxyanion hole are in gray. Also included are several active site residues. Helices are labeled successively with capital letters and ␤-strands with lowercase letters and the domain number. The active site residues are labeled with the single-letter amino acid code. (b) Representative electron density for the active site of molecule D with the covalently bound inhibitor in stereo. The electron density maps are of the form 2Fo Ϫ Fc calculated with all observed diffraction data between 15.0 and 1.9 Å and contoured at a level of 1.25␴ above the mean. The inhibitor is represented with white bonds. White crosses represent the positions of refined water molecules. The view is into the active site with the general acid–base catalyst His D44 on the left and the residues of the oxyanion hole on the right. (c) Representative electron density in the active site of molecule B. The coloring, view and the electron density are similar to those in b. Figures 1a, 2, 3, 4, and 5 were prepared with the programs MolScript and Raster-3D (Bacon and Anderson, 1988; Esnouf, 1997; Kraulis, 1991; Merritt and Murphy, 1994). 156 BERGMANN ET AL. INHIBITOR COMPLEX OF 3C PROTEINASE FROM HEPATITIS A VIRUS 157 158 BERGMANN ET AL.

FIG. 2. Stereo view of a superposition of the C␣ traces of all four molecules of the structure of HAV 3C (C24S, F82A) acetyl-Val-Phe- amide. The view is rotated 90° to the right with respect to Fig. 1a. Molecules A to D are shown in light gray to black. The position of the nucleophilic active site residue Cys 172 is marked; the C-termini and residues from the structurally most diverse regions are labeled. have some significance for the polyprotein processing in HAV. Despite the fact that residues 77 to 83 have quite different conformations in the crystal structures and it is safe to assume that this loop can have several different conformations in the molecule, the same does not apply for Asp 84. Asp 84 of HAV 3C corresponds topologically to the third member of the catalytic triad in chymotrypsin- like proteinases. However, in HAV 3C its side chain cannot interact with the general acid–base catalyst His 44 (Bergmann et al., 1997). The active sites of the four molecules of HAV 3C in the crystal structure presented here show much more conformational variability than is observed in the active sites of the chymotrypsin-like serine proteinases (Fig. 3). Nevertheless, the position of Asp 84 in all molecules from all crystal structures is the same within the accuracy of the experimental data. None of the Asp 84 side chains in any of the HAV 3C structures can interact with the general acid–base catalyst His 44, despite the inherent flexibility of the preceding loop (residues 79–83, Fig. 2). Bergmann et al. (1997) proposed that there is no catalytic triad in HAV 3C and the observed FIG. 3. Stereo views of the active site residues with the bound conformations of the Asp 84 residues in this structure inhibitors for molecule A (a), molecule B (b), and molecule D in (c). The bound inhibitor is highlighted with thicker bonds and colored in black. support that conclusion. Selected hydrogen bonds are represented by broken lines and water Residues 140 to 160 extend from two of the strands of molecules by spheres.

FIG. 4. Stereo view of a superposition of molecule D of HAV 3C (C24S, F82A) acetyl-Val-Phe-amide and the structure of an inhibitor complex of the bacterial serine proteinase SGPB (Huang et al., 1995; PDB id-code “1sgq”). The structures were superimposed using only C␣ atom coordinates of the enzymes without the inhibitor. Carbon atoms are colored in gray (SGPB) or green (HAV 3C) and the inhibitors are colored darker. The conventional coloring code is used for N (blue), O (red) and S atoms (yellow). The PЈ1 and PЈ2 residues of the two inhibitors superimpose well and bind to the respective enzymes in the same location and a similar conformation. FIG. 5. Stereo views of a model of the substrate sequence GLFSQ͉AN, that consitutes the sequence of the primary cleavage site within the polyprotein of a cell culture-adapted, fast-growing strain of HAV, bound in the active site of HAV 3C (Zhang et al., 1995). (a) The substrate sequence bound within the secondary structure elements of HAV 3C. The representation of the secondary structure and the view are similar to those in Fig. 1a. The peptide is represented as bonds with the C atoms colored in light gray. (b) Details of the binding of the substrate sequence. The substrate is colored with C atoms in light gray, O atoms in orange, and N atoms in light blue. The C atoms of the active site of HAV 3C are colored as follows: the residues of the oxyanion hole are in black, residues from ␤-strands bII2, eII, and fII and helix B (His 44) are in dark gray, residues from ␤-strand aI are in pink, residues from ␤-strand bI are in lilac, and residues from the turn that connects ␤-strands aII and bII1 are in cyan. Thicker and thinner broken lines represent hydrogen bonds with main chain and side chain atoms of the substrate sequence, respectively. INHIBITOR COMPLEX OF 3C PROTEINASE FROM HEPATITIS A VIRUS 159 the C-terminal ␤-barrel domain and contribute to one sponding residues in HAV 3C are Cys 172 (the nucleo- side of the active site (colored light gray in Fig. 1a). We phile), His 44 (the general acid–base catalyst), and have labeled this structural feature an anti-parallel ␤-rib- Pro169 to Cys172 (the oxyanion hole, dark gray in Fig. 1a; bon because it is formed by two ␤-strands that do not Bergmann et al., 1997). form part of a larger ␤-sheet or ␤-barrel, but are hydro- Molecule D of HAV 3C acetyl-Val-Phe-amide resem- gen bonded only to each other (Bergmann et al., 1997). bles the native enzyme most closely (Fig. 3c). In partic- This anti-parallel ␤-ribbon corresponds topologically to ular the conformation and the environment of the general the methionine loop in the chymotrypsin-like serine pro- acid–base residue His D44 resemble those observed in teinases. It is also present in some bacterial chymotryp- the native enzyme (Bergmann et al., 1997). A water mol- sin-like serine proteinases, such as ␣-lytic proteinase, ecule, which takes up the position of the carboxylate of where it is referred to as the ␣LP hairpin (Fujinaga et al., the third member of the catalytic triad in the structure of 1985; Sauter et al., 1998). It forms part of the specificity Bergmann et al. (1997), is also present in molecule D. pockets for the P4 and P2 residues of a substrate in the The side chain hydroxyl of Tyr D143 is within hydrogen 3C proteinases. In HAV 3C the anti-parallel ␤-ribbon is bonding distance to this water molecule. The S␥ atom of significantly longer than in other 3C proteinases (Berg- the nucleophilic Cys D172, the imidazole ring of the mann and James, 1999b). general acid–base catalyst His D44, and this water mol- In the four molecules of HAV 3C in the present struc- ecule are in a common plane. In the native enzyme the ture, the conformations of the anti-parallel ␤-ribbon are side chain of Tyr 143 interacts with, and presumably significantly different from one another and are chiefly stabilizes, this arrangement. In molecules A and B of HAV determined by the intermolecular interactions in the 3C acetyl-Val-Phe-amide, the imidazole ring of the gen- crystal. The reverse turn at the very tip of the hairpin is eral acid–base catalyst His 44 is rotated out of that plane the most flexible part, but the whole anti-parallel ␤-rib- and away from the position where it could productively bon can rotate away from the core of the molecule. This interact with the nucleophile Cys 172 (Figs. 3a and 3b). In is illustrated by the position of residue B150 in Fig. 2. In molecules A and B, Tyr 143 is part of the flexible, anti- the majority of the molecules in the crystal structures of parallel ␤-ribbon of HAV 3C (light gray in Fig. 1a) and has HAV 3C, the anti-parallel ␤-ribbon interacts with the N- moved away from the active site because of crystal terminal domain and contributes residues to the active packing interactions that involve residues 147 to 152. site (Fig. 3; Bergmann et al., 1997). In molecule B of the Therefore, Tyr143 cannot stabilize the conformation of present structure an intermolecular interaction causes the imidazole of His 44 or the water molecule that is the anti-parallel ␤-ribbon to move away from the N- hydrogen bonded to the N␦1 atom of His 44. We believe terminal domain with a hinge-like motion of about 20° that the missing interaction between Tyr 143 and His 44 (Fig. 2). As a result, there is a wide gap between the causes the nonproductive conformation of the imidazole N-terminal domain of HAV 3C and the anti-parallel ␤-rib- of His 44. This supports the notion that Tyr143 is impor- bon in molecule B. In the recently determined structure of tant for catalysis and helps to maintain the proper orien- ␣-lytic protease complexed with its prosegment, the pro- tation and possibly the protonation state of the general segment causes the ␣LP hairpin to move away from the acid–base catalyst His 44 in HAV 3C (Bergmann et al., core of the molecule (Sauter et al., 1998). The proseg- 1997). ment-induced conformational change of the ␣LP hairpin Clearly the active site of HAV 3C is much more flexible in ␣-lytic protease resembles the change in the confor- than that of the related chymotrypsin-like serine protein- mation of the anti-parallel ␤-ribbon in molecule B of the ases. What functional significance this flexibility has is present structure. This suggests that the flexibility of the less obvious. The subtle conformational differences be- anti-parallel ␤-ribbon plays a role in the autocatalytic tween the oxyanion holes of the molecules in the present excision of the 3C proteinases, similar to its role in some structure and the native enzyme could simply be due to bacterial chymotrypsin-like serine proteinases. the presence of an additional methylene group in the acetyl moiety of the inhibitor, compared to the acyl- Active site structure enzyme intermediate formed by a natural substrate. Figure 3 shows the details of the active sites of mol- Inhibitor binding and implications for substrate ecules A, B, and D with the bound acetyl-Val-Phe-amide recognition by HAV 3C inhibitors. Conformational angles of important active site residues and the distances of some interactions in the The inhibitor used in the present study was designed active site are presented in Table 3. The chemical groups to interact covalently with the enzyme and to bind spe- Ј Ј present in the active sites of proteolytic enzymes are a cifically to the S1 and S2 sites of the HAV 3C proteinase nucleophile, a general acid–base catalyst, and an elec- (Mckendrick et al., 1998). The covalent bond between the trophilic feature commonly called the oxyanion hole S␥ atom of the nucleophilic Cys172 residue and the (Brocklehurst et al., 1998; Wharton, 1998). The corre- methylene group of the acetyl portion of the inhibitor was 160 BERGMANN ET AL.

TABLE 3

Active Site Conformationsa and Enzyme Inhibitor Interactions

Ab Bb Cb Db

Pro 169 ␸ Ϫ66 Ϫ82 Ϫ72 Ϫ80 ␺ 163 160 166 163 Gly 170 ␸ 97 84 85 69 ␺ Ϫ18 11 Ϫ11 3 Cys 172 ␸ Ϫ60 Ϫ55 Ϫ58 Ϫ54 ␺ 144 135 138 162

␹1 Ϫ69 Ϫ76 Ϫ76 Ϫ74

His 44 ␹1 Ϫ35 71 70 69

␹2 58 Ϫ75 Ϫ66 Ϫ90

Asp 84 ␹1 Ϫ64 Ϫ55 3 Ϫ56

␹2 Ϫ79 Ϫ62 38 Ϫ32

Tyr 143 ␹1 Ϫ58 75 57 58

␹2 Ϫ85 Ϫ80 81 Ϫ90 30 44 48 43 Acetyl 306 ␺ 109 81 91 92

Val 307 PЈ1 ␸ Ϫ97 Ϫ63 Ϫ61 Ϫ56 ␺ 119 132 154 151

␹1 Ϫ161 178 Ϫ60 Ϫ48

Phe 308 PЈ2 ␸ Ϫ34 Ϫ59 Ϫ60 Ϫ68 ␺ 125 Ϫ54 164 128

␹1 Ϫ65 Ϫ176 Ϫ76 Ϫ69

␹2 Ϫ47 54 Ϫ40 Ϫ20 Acetyl CAO...Gly170 NH 2.67 Å (3.05 Å) 2.77 Å (3.48 Å) Acetyl CAO...Cys172 NH 2.81 Å 2.8 Å 2.82 Å 2.93 Å ... Val28 CAO Val PЈ1 NH 2.73 Å 2.69 Å 2.77 Å 2.86 Å ... c Val PЈ1 CAO Gly170 NH (3.27 Å) 3.22 Å 2.43 Å 2.89 Å His44 N⑀...Cys172S␥ (4.05 Å) 3.49 Å (4.05 Å) 3.54 Å ␦... His44 N H2O — 2.74 Å — 2.65 Å (177)d (401)d ... Tyr143 OH H2O — (8.1 Å) — 3.29 Å ... Asp84 CAO H2O — 2.99 Å — 2.87 Å

a All conformational angles in this table are in degrees. b A, B, C, and D refer to the four molecules of the complex of HAV 3C protease in the asymmetric unit. c Distances listed in parentheses are not hydrogen bonds. d Residue number of the water molecules. immediately evident in the initial electron density maps this conformation, the enzyme inhibitor interactions, and (Figs. 1b and 1c). The main chain of the dipeptide inhib- the active site conformations in the four molecules in the itor is bound between the oxyanion hole and the edge structure of HAV 3C acetyl-Val-Phe-amide are listed in strand (bI) of the N-terminal ␤-barrel domain (Fig. 1a). Table 3 and shown in Fig. 3. Figure 4 shows a superposition of molecule D of HAV 3C The peptide bond connecting the valine and phenylal- acetyl-Val-Phe-amide onto the corresponding residues in anine residues of the dipeptide inhibitor forms a hydro- the structure of the complex of the bacterial chymotryp- gen bond with a peptide carbonyl (Val 28) from the edge sin-like serine proteinase SGPB and a cognate protein strand (␤-bI) of the N-terminal ␤-barrel (Table 3; Val28 A ... Ј inhibitor (SGPB-OMTKY3 complex, PDB ID-code “1sgq,” C O Val P 1 NH). This interaction is conserved within Huang et al., 1995). enzyme substrate and enzyme inhibitor complexes of Ј Ј The positions of the P1 and P2 residues in the two chymotrypsin-like proteinases. The acetyl moiety of the structures and the interactions between the enzymes inhibitor binds in the oxyanion hole of the enzyme. Com- and the main chain atoms of the inhibitors are very pared to the substrate of a proteinase, the acetyl moiety similar. Therefore, the acetyl-Val-Phe-amide inhibitor of the inhibitor has an additional methylene group. Dur- Ј Ј does indeed bind to the S1 and S2 sites of the proteinase, ing the catalytic reaction of proteolytic enzymes the car- in the manner for which it was designed (Mckendrick et bonyl of the scissile bond forms two hydrogen bonds al., 1998) and in the canonical conformation that is ex- inside the oxyanion hole (acetyl CAO...Gly170 NH and Ј Ј A ... pected for the P1 and P2 residues of a substrate of a acetyl C O Cys172 NH). These two hydrogen bonds chymotrypsin-like proteinase (Bode and Huber, 1992; Pe- are believed to facilitate catalysis by orienting the scis- rona and Craik, 1995; Read and James, 1986). Details of sile bond for the nucleophilic attack, stabilizing the de- INHIBITOR COMPLEX OF 3C PROTEINASE FROM HEPATITIS A VIRUS 161 veloping negative charge of the tetrahedral intermediate, There are numerous potential hydrogen-bond donors and facilitating charge separation during the formation of and/or acceptors (Gln 15, Asn 30, Thr 122, Asp 124, and the new bond in the acyl-enzyme intermediate (Brockle- Gly170) that line the pocket. In molecule B a well-refined hurst et al., 1998; Wharton, 1998). The carbonyl group of water molecule is bound in place of the phenylalanine the acetyl moiety in the four molecules in the structure side chain (Figs. 1c and 3b). presented here is slightly rotated toward the nucleophilic Because of the chemical reactivity of some of the side cysteine to accommodate the additional methylene car- chains it was not possible to include all amino acid bon of the acetyl group (Table 3 and Fig. 3). As a result, residues in the study of Mckendrick et al. (1998). Lysine, Ј the oxygen of the carbonyl of the acetyl moiety of the which is the P2 residue of the primary cleavage site of inhibitor is not centered in the oxyanion hole and only the the HAV polyprotein (Martin et al., 1995; Schultheiss et hydrogen bond to the amide of Cys 172 shows the al., 1994, 1995b), could not be included. One of the amino expected geometry for a good hydrogen bond (Table 3). acid substitutions that distinguish a fast-growing, cell Ј The valine side chain in the P1 position of the inhibitor culture-adapted strain of HAV is a lysine to asparagine Ј makes nonspecific hydrophobic interactions with the mutation in the P2 position of the primary cleavage site ͉ ͉ Ј side chains of Val 28 and Met 29 of the enzyme. Both (GLFSQ AKI to GLFSQ ANI, Zhang et al., 1995). A P2 low-energy conformations of a valine side chain (gϩ ϳ asparagine residue was also not present in any of the Ϫ60.0° and t ϳ 180°, Janin et al., 1978) are observed in tested inhibitors. Modeling studies indicate that the side the inhibitor molecules of the present structure (Fig. 3, chain of an asparagine residue would be the best fit for Ј ␹ Ј Ј Table 3; Val 307 P1 1). There is no defined S1 pocket in the S2 pocket of HAV 3C. Its size would allow it to fit HAV 3C. This correlates well with the fact that compari- nicely into the pocket in its lowest side chain conforma- sons of the sequences of natural cleavage sites within tion and at the same time it could form several hydrogen the HAV polyprotein does not reveal a preference for a bonds (Fig. 5). A lysine residue would also fit well into the Ј Ј specific amino acid residue in P1 (Bergmann, 1998, and S2 pocket because its side chain is more flexible and it references therein, Seipelt et al., 1999). could donate several hydrogen bonds (Bergmann and In three of the four molecules in this structure, the side James, 1999a). Ј Ј chain of the phenylalanine residue in the P2 position is in The defined S2 pocket is a unique feature of HAV 3C. a gϩ conformation; it fits into a pocket formed between It does not exist in the structures of other picornaviral 3C the two first ␤-strands of the N-terminal domain of the proteinases (Matthews et al., 1994; Mosimann et al., enzyme, the reverse turn that connects ␤-strands aII and 1997). Therefore, and because asparagine is the best fit Ј bII1 in the C-terminal domain and the oxyanion hole for the S2 pocket of HAV 3C, we suggest that the residue Ј Ј (Figs. 1a and b). In molecule B, the side chain of the P2 in the P2 position plays a role in the differentiation of the Phe is in its second possible low-energy conformation (t) cleavage sites in the HAV polyprotein. Among HAV 3C Ј L S ͉ and does not reach into the S2 pocket of the enzyme cleavage sites with the general sequence X /IX /TQ XXX, Ј Ј (Figs. 1c and 3b). An intermolecular interaction with a the fit of the P2 residue into the corresponding S2 pocket lysine side chain from an adjacent molecule (Lys C106) of HAV 3C would be an important factor to select the presumably causes this unique conformation of the in- primary cleavage sites. According to our modeling stud- Ј hibitor side chain in molecule B. A water molecule is ies, asparagine is the best fit for the S2 pocket of HAV 3C. Ј bound in the S2 pocket of molecule B. The lowest energy conformations for the two dihedral MATERIALS AND METHODS angles, which define the side chain conformation of a ␹ ϭϪ ϩ ␹ ϭ phenylalanine residue, are 1 60° (g ) and 2 90° The double-mutant C24S, F82A of HAV 3C was purified (t) (Janin et al., 1978). Whereas the phenylalanine side from a bacterial overexpression system as previously Ј chains of the inhibitors bound in the S2 pocket of HAV 3C described (Malcolm et al., 1992). The inhibitor iodoacetyl- ␹ ϩ are close to a 1 g conformation, the aromatic ring does valyl-phenylalanylamide was synthesized as described not fit into this pocket without being rotated away from its by Mckendrick et al. (1998). Purified HAV 3C (C24S, F82A) ␹ Ј low-energy conformation. The 2 angles of the P2 phe- was incubated with a fivefold molar excess of iodoacetyl- Ј nylalanine residues, which are bound in the S2 specificity valyl-phenylalanylamide and the formation of a complex pocket, are significantly different from 90° (Table 3; was confirmed by mass spectroscopy (Mckendrick et al., Ј ␹ Phe308 P2 2). The aromatic ring of the inhibitor side 1998). The crystals of the inhibitor complex were grown chain also forces a slight rotation of the peptide bond by the hanging-drop vapor diffusion method. The hang- that connects Met 29 and Gln 30 of HAV 3C to fit into the ing drops were prepared from 2 ␮l of HAV 3C acetyl-Val- Ј S2 pocket in the enzyme. The corresponding methionine Phe-amide complex at 10 mg/ml in 10 mM Tris, pH 7.5, residues (A29, C29, and D29) are flagged as outliers in a and 6 ␮l of the reservoir solution. The hanging drops Ramachandran plot. Therefore, the phenylalanine side were equilibrated against a reservoir solution consisting Ј chain is not a perfect fit for the S2 pocket of the enzyme. of 1 ml of 100 mM Tris, pH 8.5, 5% DMSO, and 18% Ј Furthermore, the S2 pocket of HAV 3C is not hydrophobic. polyethylene glycol 8000. Crystals were transferred by 162 BERGMANN ET AL. serial transfer into a similar solution with an additional Brocklehurst, K., Watts, A. B., Patel, M., Verma, C., and Thomas, E. W. 15–20% glycerol over a period of a few minutes. Diffrac- (1998). Cysteine proteinases. In “Comprehensive Biological Cataly- tion data were collected at beam line 7-1 of the Stanford sis” (M. L. Sinnott, Ed.). Academic Press, London. Bru¨nger, A. T. (1993). “X-Plor: A system for X-ray crystallography and Synchrotron Radiation Laboratory at a crystal tempera- NMR.” Yale Univ. Press, New Haven, CT. ture of 100 K. The data were processed with the HKL data Collaborative Computational Project No. 4 (1994). The CCP4 suite: processing suite of programs (Otwinowski and Minor, Programs for protein crystallography. Acta Crystallogr. Sect. D 50, 1997) and all further calculations utilized the programs of 760–763. the CCP4 suite of crystallographic programs (Collabora- Dragovich, P. S., Webber, S. E., Babine, R. E., Fuhrman, S. A., Patick, A. K., Matthews, D. A., Lee, C. A., Reich, S. H., Prins, T. J., Marakovits, tive Computational Project No. 4, 1994). The structure J. T., Littlefield, E. S., Zhou, R., Tikhe, J., Ford, C. E., Wallace, M. B., was solved by the method of molecular replacement Meador, J. W. III, Ferre, R. A., Brown, E. L., Binford, S. L., Harr, J. E., (Rossmann, 1990) with the program package AMoRe DeLisle, D. M., and Worland, S. T. (1998a). Structure-based design, (Navaza, 1994) and using molecule A of the structure of synthesis and biological evaluation of irreversible human rhinovirus the native enzyme (Bergmann et al., 1997) as a search 3C protease inhibitors. 1. Michael acceptor structure–activity studies. J. Med. Chem. 41, 2806–2818. model. Initial refinement was performed with a version of Dragovich, P. S., Webber, S. E., Babine, R. E., Fuhrman, S. A., Patick, the program X-Plor (Bru¨nger, 1993), which had an imple- A. K., Matthews, D. A., Reich, S. H., Marakovits, J. T., Prins, T. J., Zhou, mentation of a maximum-likelihood refinement target R., Tikhe, J., Littlefield, E. S., Bleckman, T. M., Wallace, M. B., Little, (Pannu and Read, 1996). The final cycles of refinement T. L., Ford, C. E., Meador, J. W., III, Ferre, R. A., Brown, E. L., Binford, were performed with the program package TNT (Tronrud S. L., DeLisle, D. M., and Worland, S. T. (1998b). Structure-based design, synthesis and biological evaluation of irreversible human et al., 1987; Tronrud, 1992). All model building and graph- rhinovirus 3C protease inhibitors. 2. Peptide structure–activity stud- ical manipulation were performed with the program O ies. J. Med. Chem. 41, 2819–2834. (Jones et al., 1991; Jones and Kjeldgaard, 1995). The Esnouf, R. M. (1997). An extensively modified version of MolScript that figures were prepared with the programs Bobscript (Es- includes greatly enhanced coloring capabilities. J. Mol. Graph. 15, nouf, 1997; Kraulis, 1991) and Raster-3D (Bacon and 133–138. Fujinaga, M., Delbaere, L. T. J., Brayer, G. D., and James, M. N. G. (1985). Anderson, 1988; Merritt and Murphy, 1994). The coordi- Refined structure of ␣-lytic protease at 1.7Å resolution. J. Mol. Biol. nates of HAV 3C acetyl-Val-Phe-amide have been depos- 184, 479–502. ited with the Protein Data Bank (PDB ID-code “1qa7”). Gauss-Mu¨ller, V., Ju¨rgensen, D., and Deutzmann, R. (1991). Autocatalytic cleavage of recombinant 3C proteinase of hepatitis A virus. Virology ACKNOWLEDGMENTS 182, 861–864. Gorbalenya, A. E., and Snijder, E. J. (1996). Viral cysteine proteinases. We thank Annette Martin, Institut Pasteur for helpful advice and the Perspect. Drug Disc. Design 6, 64–86. staff of the Stanford Synchrotron Radiation Laboratory (SSRL) for the Graff, J., Richards, O. C., Swiderek, K. M., Davis, M. T., Rusnak, F., excellent technical support during data collection. This work was sup- Harmon, S. A., Jia, X. J., Summers, D. F., and Ehrenfeld, E. (1999). ported by the National Institute of Allergy and Infectious Diseases of Hepatitis A virus capsid protein VP1 has a heterogeneous C termi- the United States, the Medical Research Council of Canada and the nus. J. Virol. 73, 6015–6023. Alberta Heritage Foundation for Medical Research. X-ray diffraction Harmon, S. A., Updike, W., Jia, X.-Y., Summers, D. F., and Ehrenfeld, E. data were collected at beam line 7-1 of SSRL. 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