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Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protease domain

2000; Springer Nature; Volume: 19; Issue: 6 Linguagem: Inglês

10.1093/emboj/19.6.1195

1460-2075

Gaetano Barbato, Daniel O. Cicero, Florence Cordier, Frank Narjes, Benjamin Gerlach, Sonia Sambucini, Stephan Grzesiek, Victor G. Matassa, Raffaele De Francesco, Renzo Bazzo,

Monoclonal and Polyclonal Antibodies Research

Article15 March 2000free access Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protease domain G Barbato Corresponding Author G Barbato Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author D.O. Cicero D.O. Cicero Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author F Cordier F Cordier IBI-2, Forschungszentrum Jülich, Postfach 1913, Jülich, 52425 Germany Search for more papers by this author F Narjes F Narjes Department of Medicinal Chemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author B Gerlach B Gerlach Department of Medicinal Chemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author S Sambucini S Sambucini Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author S Grzesiek S Grzesiek IBI-2, Forschungszentrum Jülich, Postfach 1913, Jülich, 52425 Germany Present address: Biozentrum der Universität Basel, University of Basel, Klingelbergstrasse 70, CH-4056 Switzerland Search for more papers by this author V.G. Matassa V.G. Matassa Department of Medicinal Chemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author R. De Francesco R. De Francesco Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author R Bazzo Corresponding Author R Bazzo Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author G Barbato Corresponding Author G Barbato Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author D.O. Cicero D.O. Cicero Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author F Cordier F Cordier IBI-2, Forschungszentrum Jülich, Postfach 1913, Jülich, 52425 Germany Search for more papers by this author F Narjes F Narjes Department of Medicinal Chemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author B Gerlach B Gerlach Department of Medicinal Chemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author S Sambucini S Sambucini Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author S Grzesiek S Grzesiek IBI-2, Forschungszentrum Jülich, Postfach 1913, Jülich, 52425 Germany Present address: Biozentrum der Universität Basel, University of Basel, Klingelbergstrasse 70, CH-4056 Switzerland Search for more papers by this author V.G. Matassa V.G. Matassa Department of Medicinal Chemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author R. De Francesco R. De Francesco Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author R Bazzo Corresponding Author R Bazzo Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy Search for more papers by this author Author Information G Barbato 1, D.O. Cicero1, F Cordier2, F Narjes3, B Gerlach3, S Sambucini1, S Grzesiek2,4, V.G. Matassa3, R. De Francesco1 and R Bazzo 1 1Department of Biochemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy 2IBI-2, Forschungszentrum Jülich, Postfach 1913, Jülich, 52425 Germany 3Department of Medicinal Chemistry, IRBM ‘P.Angeletti’, Via Pontina km 30.600, 00040 Pomezia, Roma, Italy 4Present address: Biozentrum der Universität Basel, University of Basel, Klingelbergstrasse 70, CH-4056 Switzerland *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2000)19:1195-1206https://doi.org/10.1093/emboj/19.6.1195 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Few structures of viral serine proteases, those encoded by the Sindbis and Semliki Forest viruses, hepatitis C virus (HCV) and cytomegalovirus, have been reported. In the life cycle of HCV a crucial role is played by a chymotrypsin-like serine protease encoded at the N-terminus of the viral NS3 protein, the solution structure of which we present here complexed with a covalently bound reversible inhibitor. Unexpectedly, the residue in the P2 position of the inhibitor induces an effective stabilization of the catalytic His–Asp hydrogen bond, by shielding that region of the protease from the solvent. This interaction appears crucial in the activation of the enzyme catalytic machinery and represents an unprecedented observation for this family of enzymes. Our data suggest that natural substrates of this serine protease could contribute to the enzyme activation by a similar induced-fit mechanism. The high degree of similarity at the His–Asp catalytic site region between HCV NS3 and other viral serine proteases suggests that this behaviour could be a more general feature for this category of viral enzymes. Introduction The hepatitis C virus (HCV) infects ∼3% of the world population, and since it causes chronic liver disease it is considered a major health problem worldwide (World Health Organization, 1999). Patients with chronic infection can develop liver cirrhosis and are at high risk of developing hepatocellular carcinoma (Avital, 1998). Neither a vaccine against viral infection nor effective therapy has been developed to date. HCV represents the most widely spread and challenging viral infection to block. HCV is a positive-sense, single-stranded RNA virus and belongs to the Flaviviridae family. It consists of ∼9.6 kb, which in infected cells are translated into a polyprotein of ∼3011 amino acids. The genome organization comprises the structural proteins C, E1 and E2, and the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B, which are released by action of both host cell and virally encoded proteases (Neddermann et al., 1997; Bartenschlager, 1999). The N-terminal domain of the HCV NS3 protein contains a serine protease, belonging to the chymotrypsin family (Lesk and Fordham, 1996), which is responsible for the proteolytic cleavage at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A and NS5A/NS5B junctions of the viral polyprotein (Neddermann et al., 1997). The NS3 protease thus plays a pivotal role in the maturation of the viral polyprotein. Consequently, the activity of this enzyme has been studied under a broad range of conditions in view of its potential as a target for antiviral therapy (Bartenschlager, 1999; De Francesco and Steinkühler, 1999). Some enzymatic and structural features make this viral enzyme unique among the serine protease family: the serine protease domain is covalently attached to an RNA helicase possessing NTPase activity, it requires unusually long substrates (P6–P4′) for effective cleavage and possesses a solvent-accessible structural zinc-binding site (De Francesco and Steinkühler, 1999). In addition, the action of a virus-encoded protein cofactor, NS4A, is required for some but not all of the NS3-dependent proteolytic cleavage events. Thus, NS4A is necessary for the proteolytic processing of the NS4A/NS4B and NS4B/NS5A cleavage sites, whereas it only enhances the NS3 protease activity observed on the NS5A/NS5B cleavage site (Bartenschlager, 1999). NS4A functions as an activator of the NS3 serine protease by forming a non-covalent complex. The crystallographic (Love et al., 1996) and NMR solution (Barbato et al., 1999) structures of the uncomplexed enzyme, and the crystallographic structure of a complex with a peptide spanning the core domain of NS4A (Kim et al., 1996; Yan et al., 1998), have been solved. The interaction with NS4A requires the 22 N-terminal residues of NS3 and a 12-residue sequence in the centre of NS4A, which can be supplied as a synthetic peptide without loss of activation function (Bartenschlager, 1999). Comparative analysis of the crystallographic three-dimensional structures of the NS3 protease suggested a possible mechanism for the activation of the enzyme (see below). However, this model is not entirely satisfactory since it does not explain all the available biochemical data. In particular, the current model of action does not account for the proteolytic activity observed on the NS4A-independent substrates. Figure 1 schematizes the basic steps of the current general model of action of the serine protease family (Fersht, 1985; Polgar, 1989; Phillips and Fletterick, 1992). The energy implications and the precise role of each catalytic residue in this model are currently under debate (Cleland et al., 1998; Warshel, 1998). However, all authors agree that a stable network of hydrogen bonds (Figure 1A) is required for a fully active enzyme and for the nucleophilic attack that leads to the tetrahedral intermediate of Figure 1B and subsequent hydrolysis of the acyl-enzyme (Figure 1C). The NS3 mechanistic model of action proposed to date is based on the observation that, in the crystal structure obtained in the absence of NS4A, the position of the catalytic aspartate (Asp81) significantly deviates from the configuration required for proteolysis, making the formation of a hydrogen bond with the catalytic histidine (His57) impossible. Conversely, the three catalytic residues, His57, Asp81 and Ser139, acquire the canonical serine protease conformation in the crystals obtained in the presence of the NS4A cofactor. On this basis it has been proposed that binding of NS4A to the N-terminal NS3 barrel results in spatial re-organization of the serine protease catalytic triad, ultimately leading to the formation of an active enzyme (Love et al., 1998). This model has been gaining favour, as documented by a recent review (Bartenschlager, 1999). While interesting, this model does not explain how the NS3 protease can be active on substrates such as the NS5A/NS5B junction in the absence of NS4A. A model where the uncomplexed NS3 serine protease may work via the formation of a catalytic ‘dyad’ is not supported by cell culture experiments; it has in fact been shown that mutation of each single residue of the catalytic triad results in a totally uncleaved polyprotein (Hijikata et al., 1993). Thus, if in the absence of NS4A the Asp81 is not participating in the catalytic triad, as suggested by the model, the NS3 protease should be an inactive enzyme. Figure 1.Schematic representation of the general mechanism of action of serine proteases. (A) Hydrogen bond network needed for a fully active enzyme. The current mechanistic model proposes that the histidine acts as a general base during catalysis, accepting a proton from the serine as it forms a bond with the substrate carbonyl carbon, giving rise to a tetrahedral intermediate (B). The negative charge on the oxygen atom is stabilized by the oxyanion hole formed by the amide protons of the catalytic serine and of a glycine residue two positions before the serine. The H-bond between the histidine δ-NH and the aspartate carboxyl groups ensures that the imidazole ring is in the correct tautomeric form to accept the serine proton. (C) The final step of the catalysis proceeds through hydrolysis of the acyl-enzyme by a water molecule. Note that for the substrate the nomenclature of Schecter and Berger 1967) is used in designating the cleavage sites as Pn-Pn−1-P1-P1′-Pn−1′-Pn′ with the scissile bond between P1 and P1′ and the C-terminus of the substrate on the prime site. Download figure Download PowerPoint Recently, the solution structure of the NS3 protease has been published (Barbato et al., 1999), where the relative position of the catalytic triad, even in the absence of the NS4A cofactor, is compatible with that of a fully active serine protease. The disagreement between the crystal and solution structures is likely to be due to the fact that the former lacks the extremely important helix α3 (Figure 2B), probably due to distortions induced by crystal packing forces. This helix, present in solution, is crucial for the correct packing of the strand F1 that positions the catalytic Asp81. The NMR evidence therefore suggests that NS4A is likely to play a different and more subtle role with respect to NS3 serine protease activation. The solution structure of the free enzyme, however, also poses a new question, in fact the hydrogen bond between the carboxylate group of Asp81 and the δ-NH of the His57 is not stable since the whole region is completely solvent exposed (Barbato et al., 1999). This is also reflected in the unusually high temperature factors of the backbone atoms of the loop E1–F1 (including Asp81) in the crystallographic structures published to date, also in the presence of NS4A, indicating an intrinsic mobility at this site. A stable network of hydrogen bonds as schematized in Figure 1A is, however, essential in order to have an active enzyme. In all chymotrypsin-like enzymes this is ensured by burying the Asp–His side chain hydrogen bond and making it solvent inaccessible (McGrath et al., 1992). Thus, for NS3, either with or without the activating NS4A cofactor, one has to raise the issue of how the Asp–His hydrogen bond can be stabilized in a region that is solvent accessible and affected by mobility. Figure 2.(A) Stereoview of the 20 minimum energy structures. The superposition has been obtained using the structurally conserved regions (SCRs) as defined previously in Barbato et al. (1999). The inhibitor α-C atoms are shown in red. The N-terminal 21 residues were not included in the structure calculations, since their conformation is ill defined. (B) Molscript (Kraulis, 1991) representation of the NS3 protease domain, the chymotrypsin-like common SCRs are coloured in blue and the structural element labels follow the chymotrypsin-like nomenclature. The residues forming the secondary structural elements are: strands A1 (34–37), B1 (41–44), C1 (51–55), D1 (63–66), E1 (69–78), F1 (82–86), A2 (104–108), B2 (122–126), C2 (141–145), D2 (150–152), E2 (155–159), F2 (166–170); helices α1 (55–59), α2 (132–136), α3 (173–180). Download figure Download PowerPoint In this work we present the first structure of the complex of the NS3 protease domain with a covalently bound α-ketoacid peptidic inhibitor (see inset in Figure 3). Since the NS3 protease is involved in the maturation process of the viral polyprotein, this structure is relevant for the design of novel therapeutic agents active against HCV. The inhibitor used is a second generation inhibitor, which is based on the previous optimization of product inhibitors (Ingallinella et al., 1998). It has already been shown that the mode of binding of P region-derived product inhibitors is similar to the ground state binding of the corresponding substrates (Ingallinella et al., 1998). The C-terminal ketoacidic moiety only provides additional binding energy (Narjes et al., 2000). The details of the NS3–inhibitor complex structure are then used as a tool to gain information also on the substrate interaction itself. Our findings support a plausible role of the substrate itself in enzyme activation. The mechanism of substrate-induced protease activation provides a framework to explain how the NS3 protease could at least be partially active even in the absence of the NS4A cofactor. Thus, we propose a new model of mechanistic action for the NS3 protease that we believe reconciles the available experimental evidence presented to date. This mechanism of substrate-induced enzyme activation appears to be unique among the chymotrypsin-like proteases. Figure 3.The inset shows the reversible covalently bound inhibitor α-ketoacid Boc-Glu-Leu-(γdi-fluoro)Abu; the corresponding positions in the substrate-like nomenclature (P1, P2 and P3) are also indicated. The asterisk labels the activated carbonyl moiety, which acts as binding group and becomes the hemiketal chiral carbon upon complex formation. The spectra show regions of the 1D 13C experiment where the hemiketal carbon of the complex resonates. At pH 6.6 (bottom) only one doublet resonance is visible at 102.6 p.p.m., corresponding to the S configuration; at pH 5.5 (top), two resonances of similar intensity appear at 102.6 and 97.6 p.p.m. for the R and S configurations, respectively. The hemiketal signals are doublets since they are coupled with the α-C, which is also 13C labelled, while the carboxyl atom is unlabelled. The intense singlet at ∼105 p.p.m. represents a buffer resonance. Download figure Download PowerPoint Results and discussion NS3 protease overall topology In Figure 2A a stereoview of the backbone bundle is presented. The structures were calculated excluding the first 21 residues, which, as in the case of the free enzyme (Barbato et al., 1999), are mobile and ill defined in solution (the constraints database and the statistical analysis of the structure quality are reported in Table I). Structurally conserved regions (SCR) that are common to all the known chymotrypsin-like proteases (Greer, 1990) are very well conserved in the complex (Figure 2B). NS3 protease is a relatively small protein (180 residues) and belongs to the sub-class of small chymotrypsin-like proteases (Bazan and Fletterick, 1988). As such it makes an economical use of loops, since it lacks a series of connecting elongations that are a common feature of the longer cellular proteases. The solution structure of the complex is similar to the solution structure of the free enzyme (Barbato et al., 1999), with a root mean square deviation (r.m.s.d.) of 1.05 Å for the SCR residues of the minimized average structures. The significant differences are located at the catalytic triad and in the C-terminal domain S-site where the inhibitor is bound (Figure 2A). The direct participation of the activating cofactor NS4A in the recognition or binding of the S-site inhibitors can be ruled out on structural grounds: NS4A binds at the N-terminal barrel, remote from the region of interest, and does not appear to affect the conformation at the active site directly. This conclusion is in agreement with the previously reported mapping of the interaction with substrate-derived inhibitors (Cicero et al., 1999), and is also confirmed by comparison with the crystal structure of the NS3/NS4A–inhibitor ternary complex (S.Di Marco and M.Sollazzo, personal communication). Table 1. Experimental restraints and structural statistics NMR constraints NOE total 3023 intra 905 inter short distance ( i + 3) 1132 Generic total 86 H-bond 81 Zn-binding site 5 Dihedral total 177 φ 91 χ1 86 Stereospecific methylene groups 92/219 methyl groups 62/68 distance (Å) 0.056 ± 0.002 dihedral(°) 3.499 ± 0.642 Deviations from idealized geometry bonds (Å) 0.005 ± 0.0002 angles (°) 0.812 ± 0.015 impropers (°) 1.461 ± 0.008 Coordinates precision referred to mean structure (Å) residues SCR + helicesb backbone 0.487 ± 0.111 all heavy atoms 0.860 ± 1.161 all residuesc backbone 0.738 ± 0.124 all heavy atoms 1.120 ± 0.191 Ramachandran analysisd % of residues in most favoured regions 71.8 ± 2.6 % of residues in allowed regions 21.5 ± 2.9 % of residues in generously allowed regions 4.5 ± 1.3 % of residues in disallowed regions 2.2 ± 1.4 a None of the structures exhibited distance violations >0.5 Å or dihedral angle violations >5°. b Residues: 34–37, 41–44, 51–59, 63–66, 69–78, 82–86, 104–108, 122–126, 131–145, 150–152, 155–159, 166–170, 173–180. c Residues: 33–95, 104–180, 188–190. d The program PROCHECK (Laskowski et al., 1993) was used to assess the overall quality of the structures. All the residues 22–186 and the inhibitor 188–190 (in total 167 residues) were used for the Ramachandran plot statistical evaluations. Inhibitor binding stereochemistry The carbon of the activated keto-group (see Figure 3, inset) of the inhibitor becomes a hemiketal quaternary carbon upon binding to the γ-O of the catalytic serine. Both R and S configurations are possible at this chiral centre. By using a sample with a selectively labelled 13C quaternary carbon, we could observe that in the pH interval 5.3–5.7 both chiral forms are present together with a small amount of the ketoacid form (non-covalently bound), whereas at pH values >6.0 only one configuration is dominant (Figure 3). This behaviour has already been observed by NMR on the complexes of chymotrypsin–N-acetyl-phenylalaninal (Shah et al., 1984) and trypsin–leupeptin (Ortiz et al., 1991). The X-ray structure of the Streptomyces griseus protease A bound to chymo-statin (pH 4.1) showed the simultaneous presence of both configurations (Delbaere and Brayer, 1985). Although our structural data (collected at pH 6.6) do not allow an unambiguous assignment of the stereochemistry, cogent arguments are presented below in favour of the S hemiketal carbon configuration being dominant at physiological pH. We did, however, perform structural calculations in parallel for both configurations (Figure 4A and B). Figure 4.(A and B) Selected region of the minimized average structure of the set of NMR structures assuming the R or S configuration at the hemiketal quaternary carbon, respectively. The protein backbone is shown in magenta ribbon representation, the inhibitor is represented in ball-and-stick representation with carbons in green. The relevant protein residues involved in the interaction are in stick representation with carbons in dark grey. The three positively charged residues (Arg109, Lys136 and Arg155) surrounding the catalytic site are shown and labelled. At the bottom of the figure a schematic representation of the covalent bond and the inhibitor interaction with the oxyanion hole is presented. Download figure Download PowerPoint Inhibitor binding site: P1 An expanded view of the inhibitor-bound structure, for the hemiketal carbon R and S configurations, respectively (Figure 4A and B), reveals that the specificity pocket is occupied by the di-fluoro-Abu side chain, with the γ-H proximal to Phe154. In fact the γ-H experiences a downfield shift (Δδ = 0.3 p.p.m.), which may be the result of its proximity to the deshielding zone of the Phe154 aromatic ring. The positioning of the fluorine atoms was obtained from 1H-19F NOE data. The refined structures assuming the R or S configuration appear very similar (r.m.s.d. = 0.11 Å for the averaged minimized structures). As noted also by others (Delbaere and Brayer, 1985) the hemiketal complexation causes remarkably little movement in the positions of the catalytic residues. For the R configuration (Figure 4A) the carboxyl group is oriented towards the His57 and is solvent exposed, while the hemiketal oxygen O1 is involved in H-bonds with the oxyanion hole amide groups of Ser139 and Gly137 (dHN−O = 2.5 and 3.2 Å, respectively). The R configuration, with the carboxylate moiety directed towards the His57 ring, is likely to be favoured by the protonation of the imidazole ring, which takes place below pH 5.8, as clearly shown by pH titration data (Figure 7C). Also, if the R configuration were stable at high pH, the hemiketal oxygen O1 (Figure 4A) would exhibit a lowered pKa value, since its acidic form is stabilized by the interactions in the oxyanion hole, whereas no titration effect has been observed up to pH 9.5. In the case of the S configuration (Figure 4B), the carboxylate group points towards the oxyanion hole and forms direct H-bonds with HN Ser139 and Gly137 (dHN−O = 2.5 and 2.6 Å, respectively), while the hemiketal oxygen O1 is oriented towards the His57 and is solvent exposed. In the S configuration the carboxylate in the oxyanion hole is very close to the H donor groups, which accounts for the similar and large (>2 p.p.m.) downfield shifts observed for both NH protons. Thus, on the basis of all the previous direct and indirect evidence, one can argue that the hemiketal carbon at pH 6.6 adopts the S configuration, as illustrated in Figure 4B. Figure 5.(A) Expanded view of the pairwise superposition of the NS3–inhibitor complex S configuration (average minimized structure backbone in magenta ribbon, the catalytic triad in stick representation and the ketoacid inhibitor in ball-and-stick representation with atom type colour code) and APPA–trypsin complex (PDB accession code 1tpp, backbone in black ribbon and inhibitor in black stick representation), the SCR residues' backbone atoms have been used for the superposition. The resulting distances between equivalent nitrogen atoms forming the oxyanion hole are shown. (B) Pairwise super- position of the average minimized structures of the free enzyme, blue (PDB accession code 1bt7), and the complex, assuming the S configuration, magenta, with the inhibitor shown in ball-and-stick representation (green). The catalytic triad is reported in stick representation. The HN groups forming the oxyanion hole and their distances in the two structures are shown. The SCR strands that account mostly for the total r.m.s.d. observed (1.05 Å) are A2 and C2. (C) Pairwise superposition of the minimized average structure of the set assuming the S configuration, with inhibitor I in cyan stick representation (product of reaction inhibitor complex) (Cicero et al., 1999). The backbone heavy atoms of the P1–P3 residues have been used in the superposition. Download figure Download PowerPoint Figure 6.(A and B) View of the surface of the catalytic triad (red) for the free (A) and complex (B) enzyme structures exposed to the solvent. The protein surface is shown in grey (A and B) and the inhibitor in yellow (B). Download figure Download PowerPoint Figure 7. Download figure Download PowerPoint We further observe that the positions of the residues forming the oxyanion hole (Gly137 and Ser139) are essentially invariant in the free enzyme (Barbato et al., 1999) and in the complexes; dNH Ser139 = 0.32 Å and Gly137 = 0.74 Å (r.m.s.d. of the α-C of residues 136–140 = 0.25 Å) (Figure 5B). The structure of an enzyme-bound, non-covalent inhibitor (inhibitor I) was obtained with transfer NOEs (Cicero et al., 1999), and in Figure 5C its superposition with the covalently bound ketoacid inhibitor structure (S configuration) is shown. The r.m.s.d. for the P1–P3 backbone heavy atoms' superposition is 0.22 Å. Furthermore, as shown in Figure 5C, the carboxylic end of inhibitor I is only partially stabilized by the oxyanion hole interactions since the hydrogen bond distances are longer. This characteristic is probably also reflected by the low affinity observed in the inhibitor I type family of compounds (Ingallinella et al., 1998). Comparing the S configuration of the NS3–ketoacid complex (Figure 4B) with the tetrahedral intermediate (Figure 1B), it can be seen that the ketoacid, due to its one-carbon homologated chain, is able to place its carboxyl group more effectively in the oxyanion hole. As a result the required interactions can take place without any apparent need for rearrangement. These results substantially confirm that the ketoacidic moiety's role is to add binding energy without altering the general mechanism of the interaction. Inhibitor binding site: P2 A very interesting feature is observed at the P2 Leu side chain. This side chain is positioned above the His57 imidazole moiety and also partially shields the Asp81 carboxylate moiety from the solvent (Figure 4B). The local hydrophobicity is further enhanced by the packing of the side chain methyl of Ala156 and the side chain methylenes of Arg155 in extended conformation and parallel to an ideal axis formed by the δ-NH of His57 and the COO− of Asp81. While the total exposed surface in this region is 86 Å2 in the free enzyme (Figure 6A), in the complex it is reduced to 34 Å2 (Figure 6B). For chymotrypsin-type enzymes the corresponding average value is ∼40 Å2, thus very close to the value found in the NS3–inhibitor complex. The solvent-exposed nature of this region in the free NS3 enzyme prevented the NMR detection of the signal of the histidine δ-NH hydrogen bonded to the aspartate carboxyl even at extreme temperature conditions (−8°C) (Barbato et al., 1999). This signal is commonly observed in other serine proteases (Markley, 1978; Bachovchin, 1985). The observation of this signal is an indication of the intact hydrogen bond between the catalytic Asp–His residues. It has been shown that this hydrogen bond becomes unstable when solvent exposed (Frey et al., 1994) and, on the contrary, is extremely stable when sheltered from the solvent (ΔG″ >10 kcal/mol) (Markley and Westler, 1996). The exclusion of solvent at this site upon complex formation allows the experimental observation of the signal arising from the δ-NH proton of His57. Figure 7A shows the typical downfield HN signal (1H, 14.9 p.p.m.; 15N, 180.9 p.p.m.) arising from the δ-NH of His57 (pH = 6.7, T = 288 K). Figure 7B shows the long range HSQC 1H-15N connectivities between the ϵ-CH (1H, 8.08 p.p.m.) and the δ-NH (15N, 180.9 p.p.