Conserved mode of peptidomimetic inhibition and substrate recognition of human cytomegalovirus protease

articles

nature structural biology • volume 5 number 9 • september 1998 819

1Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road / P.O. Box 368, Ridgefield, Connecticut 06877, USA. 2Boehringer Ingelheim (Canada) Ltd., Bio-MégaResearch Division, 2100 rue Cunard, Laval, Québec, Canada H7S 2G5. 3Present address: Department of Biological Sciences, Columbia University, New York, New York10027, USA.

Correspondence should be addressed to L.T. email: [email protected]

Human cytomegalovirus (HCMV), a herpesvirus, infects up to70% of the general population and can cause severe health prob-lems in immuno-suppressed individuals (organ transplant recipi-ents and AIDS patients)1. The herpesvirus protease is essential forthe production of infectious virions and is therefore an attractivetarget for the development of anti-herpes agents2. Herpesvirus pro-tease is a serine protease, but with no detectable sequence homolo-gy to other serine proteases2. The crystal structures of herpesvirusprotease enzymes in the absence of substrate or inhibitors showthat they have a unique polypeptide backbone fold3–7. The catalytictriad of herpesvirus protease is formed by the nucleophile Ser 132(HCMV protease numbering), the second member His 63, and anovel third member His 157.

Herpesvirus protease catalyzes the maturational processing ofthe herpesvirus assembly protein near its C-terminus (M-site)2. Inaddition, it is responsible for the cleavage at the R-site which releas-es the catalytically-active portion from the full-length product ofthe protease gene. The protease prefers small residues at the P1

(Ala) and P1' positions (Ser)2,8. A peptidomimetic inhibitor BILC821 (Fig. 1) was developed based on the substrate preference ofHCMV protease at the M- and the R-sites9. It inhibits the enzymewith an IC50 value of 0.3 µM. It covers the P4 to P1 position, and thebenzyl capping moiety at the C-terminus is referred loosely as theP1' group here. A para-iodo substituent on this phenyl ring (Fig. 1)was introduced for crystallographic purposes. The inhibitor con-tains an activated carbonyl in an α-ketoamide moiety as the active-site binding group. It is expected that the active-site serine residueof HCMV protease can attack this activated carbonyl and form a(reversible) covalent complex with the inhibitor.

The oxyanion hole, which would stabilize the tetrahedral transi-tion-state intermediate of the hydrolysis reaction, had been tenta-tively identified based on the crystal structure of the freeenzyme3,5,6. However, the binding modes of substrates andinhibitors were difficult to predict based on the free enzyme struc-tures6. Moreover, ~20% of the residues of the protease were found

to be disordered in the free enzyme structures. Some of theseresidues could be located near the active site of the enzyme andtherefore may participate in the binding of substrates andinhibitors. Here we present the crystal structure of HCMV proteasein complex with the peptidomimetic inhibitor BILC 821 and showits detailed interactions with the protease.

Overall structureThe crystal structure of HCMV protease (A143Q, T181M andL229M triple mutant)3 in complex with the peptidomimeticinhibitor BILC 821 has been determined at 2.7 Å resolution. Thecurrent structure model contains residues 4–46, 53–143, 152–200and 210–256 for each of four unique molecules of the protease inthe crystal. There is one inhibitor molecule (with its residues num-bered 261–265) bound to each protease monomer. No solvent mol-ecules were included in the atomic model. The R-factor is 22.6% for27,152 reflections between 6.0 and 2.7 Å resolution (92% complete).The free Rfree

10, for 7.5% of the reflections, is 33.2% (Table 1).The four unique molecules of HCMV protease in the crystal

belong to two separate dimers. The four monomers, and their asso-ciated inhibitors, have essentially the same conformation. Ther.m.s. difference in equivalent Cα positions between any pair of themonomers is roughly 0.2 Å for all the residues. The organization ofthe two dimers are very similar as well, with a r.m.s. difference inequivalent Cα positions between them of 0.3 Å.

Conformational differencesThere are significant differences both in the conformation of theprotease monomer and in the organization of the protease dimerbetween this inhibitor complex and the free enzyme reported ear-lier by us3. More residues of the protease are ordered in the cur-rent structure (Figs 2, 3), including: residues 4–9 at theN-terminus of the enzyme, which form a small helix (namedαN); residues 23–33 (loop L2)3; and residues 136–143 and 151–154(loop L9). In the free enzyme structure, residues 23–33 are disor-

Conserved mode of peptidomimetic inhibitionand substrate recognition of humancytomegalovirus proteaseLiang Tong1,3, Chungeng Qian1, Marie-Josée Massariol2, Robert Déziel2, Christiane Yoakim2 and Lisette Lagacé2

Human cytomegalovirus (HCMV) protease belongs to a new class of serine proteases, with a unique polypeptidebackbone fold. The crystal structure of the protease in complex with a peptidomimetic inhibitor (based on thenatural substrates and covering the P4 to P1' positions) has been determined at 2.7 Å resolution. The inhibitor isbound in an extended conformation, forming an anti-parallel β-sheet with the protease. The P3 and P1 side chainsare less accessible to solvent, whereas the P4 and P2 side chains are more exposed. The inhibitor binding modeshows significant similarity to those observed for peptidomimetic inhibitors or substrates of other classes of serineproteases (chymotrypsin and subtilisin). HCMV protease therefore represents example of convergent evolution. Inaddition, large conformational differences relative to the structure of the free enzyme are observed, which may beimportant for inhibitor binding.

© 1998 Nature America Inc. • http://structbio.nature.com©

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820 nature structural biology • volume 5 number 9 • september 1998

dered in one monomer, while they are stabilized by crystal packingand their conformation identified in the other monomer. In thecurrent structure, loop L2 is ordered in both monomers and has aconformation very different from that found in the free enzyme.The distances between equivalent Cα atoms of residues 27–31exceed 14 Å between the two structures. The residues in their newpositions are located near the inhibitor. Consequently this newconformation may be required for inhibitor binding.

Similarly, residues 136–143 in loop L9 probably became moreordered due to inhibitor binding, as residues 136–137 are locatednear the inhibitor. In addition, a conformational difference inresidues 134 and 135 is observed (Fig. 3), although Ser 135 is notfully ordered in the free enzyme structure. There is a steric clashbetween the inhibitor and residues 134 and 135 in their freeenzyme conformation. The positions of these residues in anotherof the free enzyme structures6 would also probably clash with theinhibitor at this position. This may explain the large differencesbetween the inhibitor binding mode observed in this study and thatmodeled based on the free enzyme structure6.

In addition to a greater number of residues being ordered in theprotease–inhibitor complex, there are large conformational differ-ences relative to the free enzyme in other regions of the protease,including residues 35–45 in helix αA, 165–169 in loop L10, 217–230in helix αF, and 231–232 in loop L15. A large conformational differ-ence is seen for the side chain of Arg 167, which binds a sulfate ion inthe free enzyme structure3. In the current structure the position ofthe sulfate ion is occupied by residues 25–27 from loop L2.

The conformational difference in helix αF and the following loopL15 may be related to the difference in the organization of the pro-tease dimer (Fig. 3). If one monomer of the protease is superimposedbetween the free enzyme and the inhibitor complex, a rotation of 6.5°around an axis mostly perpendicular to the dimer two-fold axis isneeded to bring the other monomer into an overlapping position.The cause of this change in the dimer organization is currentlyunknown. Treatment of free enzyme crystals with Na2SO4 also pro-duced a re-organization of the dimer (and a significant improvementin diffraction quality)3,11. It appears that the herpesvirus proteasedimer interface is capable of adopting alternative conformations, asthe varicellar-zoster virus protease (which is a member of the herpes-virus family) shows an even larger reorganization7.

A conformational change in HCMV protease due to the bindingof peptidomimetic inhibitors has been proposed based onobserved changes in fluorescence of the enzyme12. These fluores-cence changes have been ascribed to the change of environmentaround Trp 42 (helix αA) in the protease–inhibitor complex. Thisobservation is consistent with the present structure, in which helixαA shows a large conformational difference compared with the freeenzyme. This helix interacts with residues in loop L2 (in the com-plex) and residues 140–143 in loop L9. The Trp 42 residue is most-ly exposed in the structure of the free enzyme3, whereas it isshielded from solvent by residues Arg 136 and Pro 154 from loopL9 in the protease–inhibitor complex. Therefore, the observed flu-orescence change is probably a reflection of the ordering of loop L9upon the binding of peptidomimetic inhibitors. It is not knownwhether the new conformation of loop L2 and other segments ofthe protease, as observed in the current structure, has any effect onthe fluorescence.

Inhibitor–protease interactionsThe peptidomimetic inhibitor BILC 821 is bound in an extendedconformation and forms an anti-parallel β-sheet with the protease(Fig. 2). Three hydrogen-bonds are made between the main chainatoms of the inhibitor and strand β5 of the protease (Fig. 4), whichalso contains the catalytic Ser 132 residue. Two of the hydrogenbonds are between the P3 residue and Ser 135. The third hydrogenbond is between the main chain amide of the P1 residue and the car-bonyl group of Leu 133. A twist in strand β5 near this residue, prob-ably required by the formation of the β-barrel core of the enzyme,disallows the hydrogen bond from the P1 carbonyl oxygen to themain chain amide of Leu 133. This could facilitate the P1 residue ofthe substrate adopting a conformation suitable for catalysis.

In the complex, the side chain of Ser 132 forms a covalent bondwith the carbon atom from the activated carbonyl of the inhibitor,and the resulting oxyanion is hydrogen bonded to the main chainamide of Arg 165, consistent with predictions based on the struc-ture of the free enzyme. The side chain guanidinium group of thisArg residue, which is flexible and mostly exposed to solvent in thefree enzyme structure, is hydrogen bonded to the main chain car-bonyl oxygen of the P2 residue (Fig. 4). This may explain why Arg165 is strictly conserved among all herpesvirus proteases2.

Fig. 1 a, Chemical structure of the peptidomimetic inhibitorBILC 821 of HCMV protease. The residues of the inhibitor (P4

through P1' ) are labeled. b, Electron density at 2.7 Å resolu-tion for the BILC 821 inhibitor after four-fold NCS averagingover the two dimers. The phase information was based on thestructure of the protease only. The contour level is at 1σ.

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The extended conformation observed here matches well withthat determined by transfer NOE method for a related peptidylmethyl ketone inhibitor13, and especially with those of N-terminalpeptide substrates14. This suggests that the bound conformationobserved for this peptidomimetic inhibitor is likely to be a goodmimic for the bound conformation of the natural substrate ofHCMV protease.

The side chains of the inhibitor at the P4 and P2 positions are situated over the surface of the protease and are mostly exposed tothe solvent (Fig. 5; Table 2). The P2 side chain lies over that of His63, at a distance of ~3.5 Å. There is a hydrogen bond between thecarbonyl oxygen of the P2 side chain and the hydroxyl of Ser 144.The side chains of the P3 and P1 residues are less accessible to thesolvent. The conformational differences of residues in loops L2 andL9 introduce a significant change in the surface topology near theactive site of the enzyme (Fig. 5). These residues in their new posi-tions participate in the formation of the S1 and S3 pockets. Thesebinding pockets are clearly visible in the protease–inhibitor com-plex, but are absent in the free enzyme structure (Fig. 5).

The S1 and S3 binding pockets are mostly connected, forming alarger depression on the surface of the protease (Fig. 5). The S1 por-tion of the pocket is formed by residue Leu 32 from loop L2,

Ser 132, Leu 133, Arg 165 and Arg 166 (Table 2). This pock-et is fairly small (Fig. 5), thus explaining the preference ofherpesvirus proteases for Ala at the P1 position. Leu 133 isconserved among all herpesvirus proteases, Leu 32 is con-served in all except for infectious laryngotracheitis virus(where it is a Tyr)2.

The S3 portion of this pocket is formed by the salt bridgesamong residues Glu 31 from loop L2, Arg 137 from loop L9and Arg 165, and by residues Ser 135 and Arg 166.Therefore, the S3 pocket appears to be rather hydrophilic innature. The side chain of Arg 166 is located at the base ofthis surface depression and is rendered mostly inaccessibleto solvent by the binding of the inhibitor. The guanidiniumgroup of the side chain is near the main chain carbonyloxygen of Leu 32 and the side chain hydroxyl of Ser 135,although they are not positioned for optimal hydrogenbonding based on the current atomic model. No acidic sidechains are located nearby to balance the expected positivecharge of the guanidinium group. This Arg 166 residue isstrictly conserved among all herpesvirus proteases2.

Residues Glu 31 and Arg 137 are highly conserved among the her-pesvirus proteases, except for those from γ-herpesviruses andmurine CMV2.

The amino acid sequences near the R- and the M-sites, which arethe natural substrates, are generally conserved among herpesvirusproteases2. The core cleavage sequence covers residues P4 to P1'(Table 2). The binding pockets revealed in the protease–inhibitorcomplex are generally in agreement with the substrate preferencesof the protease, providing further support to the notion that theobserved bound conformation of the inhibitor is similar to that ofthe substrate. The substrate preferences at the R-site show consis-tent differences to those at the M-site (Table 2). The R-site residuesare not only substrates of the enzyme, but also form the C-termi-nus of the enzyme (residues 253–256). The crystal structures of thefree enzyme and the protease–inhibitor complex show that theseC-terminal residues are buried in a region away from the active site(Fig. 2) and involved in important interactions with the rest of theenzyme. The observed difference between the R-site and M-siteresidue preferences is probably due to this additional requirementon the R-site residues3. For example, HCMV protease containing amutated Tyr 253 residue (the P4 residue of the R-site) has signifi-cantly weaker catalytic activity2.

Fig. 2 a, Schematic drawing30 of the structureof HCMV protease in complex with the inhibitorBILC 821. The β-strands of the protease areshown as arrowed ribbons (in cyan), the α-helices in yellow and the connecting loops inpurple. The active site residues (Ser 132, His 63and His 157) are also shown (in gray for carbonatoms, red for oxygen, blue for nitrogen, andyellow for iodine) and labeled (red numbers).The secondary structure elements are named asin the free enzyme3. The new helix identified atthe N-terminus is named αN. The main chain ofthe inhibitor is shown as an arrowed ribbon (ingreen), and the side chain atoms are shown asstick models (in green for carbon atoms).b, Topological drawing of the backbone fold ofHCMV protease. Strand β3 appears twice in thisdrawing. The loops are numbered starting fromthe N-terminus. The inhibitor molecule is alsoshown (in thick lines).

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The current structure does not contain a true P1' residue. By com-parison with other serine proteases, the binding mode of the P1'residue and HCMV protease residues forming the S1' pocket couldbe tentatively identified. The S1' pocket includes the strictly con-served Cys 161 in HCMV protease (Table 2)2. The serine side chain


of the P1' residue can probably form a hydro-gen bond with the side chain of residue 62,which is Asp in α-, Asn in β-, and Glu in γ-her-pesvirus proteases respectively 2.

The crystal structure of the protease–inhibitorcomplex explains many of the observed structure-activity relationships for pep-tidomimetic inhibitors of HCMV protease9.N-Methylation experiments showed that themain chain amido nitrogen atoms of the P1 andP3 residues are required for activity, in agree-ment with their involvement in hydrogenbonding to the protease (Fig. 4). Only methyland ethyl groups are tolerated as P1 side chains,consistent with the presence of a small S1 pock-et. As expected from the structure, large vari-eties of substitutions at P4 and P2 are permitted(Table 2). Varying the size of the P3 side chainfrom ethyl to adamantyl had little effect (withinfive-fold) on the potency of the inhibitor. TheS3 pocket observed in the protease–inhibitorcomplex is rather large, although it probablycannot accommodate the adamantyl groupwithout some conformational re-adjustments.The crystal structure also shows the presence ofArg 166 near the bottom of this ratherhydrophilic binding pocket, prompting thesynthesis of inhibitors containing Asp, Glu and

other acidic side chains at P3. Surprisingly, these inhibitors havedramatically reduced activity against HCMV protease. It appearsthat the protease prefers a hydrophobic residue at this position,both in inhibitors and in substrates (Table 2). Further studies willbe needed to characterize the interactions in the S3 binding pocket.

Fig. 3 a, Superposition of the Cα trace of HCMVprotease in complex with BILC 821 (cyan) and thefree enzyme (yellow)3. The purple arrows, labeledwith the secondary structure elements, point toregions of large conformational differencesbetween the two structures. The inhibitor mole-cule is shown in green (for carbon atoms). Note thesteric clash between residues 134–135 in the freeenzyme structure and the P2 side chain of theinhibitor. b, Superposition of the dimer of HCMVprotease in the free enzyme state (yellow) and theinhibitor complex (cyan). The structure of onemonomer was used for the superposition (shownat the bottom). The inhibitor bound to thismonomer is shown in green (for carbon atoms).The two-fold axis of the dimer is indicated with thethick purple line.

Fig. 4 Schematic drawing showing the interactions between the mainchain atoms of the BILC 821 inhibitor and HCMV protease. Possiblehydrogen-bonds are shown as dashed lines. The bond between the sidechain of Ser 132 and the activated carbonyl of the inhibitor is also indi-cated. The hydrogen bond from the oxyanion to the main chain amidonitrogen of Arg 165 is not shown.

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Other serine proteasesDespite the unique backbone fold of HCMV protease, the catalytictriad is organized in a similar fashion as that found in other classesof serine proteases, such as chymotrypsin and subtilisin3–6. Thestructure of the HCMV protease–inhibitor complex allows adetailed comparison of the binding modes of inhibitors (or sub-strates) to the various serine proteases. A least-squares superposi-tion between HCMV protease in complex with BILC 821 andchymotrypsin in complex with the turkey ovomucoid thirddomain (PDB entry 1CHO)15 was performed using the side chainsof the second-member His residue, the Cα and Cβ atoms of thecatalytic serine residue, and the Cα atoms of the P1 and P2 residuesof the inhibitors: the r.m.s. difference for the 10 atom pairs is 0.4 Å.A similar overlap between HCMV protease and subtilisin Carlsbergin complex with eglin c (PDB entry 1CSE)16, including the Cαatoms of the P3 residues of the inhibitors as well, produced an r.m.s.difference of 0.4 Å for the 11 atom pairs.

The bound conformation of the inhibitors for the P2 and P1

residues are remarkably similar among HCMV protease, chy-motrypsin and subtilisin (Fig. 6). The inhibitors for all threeenzymes form an anti-parallel β-sheet with the protease, with asimilar hydrogen bonding pattern between the inhibitor and theprotease (Fig. 6)17. There are three conserved hydrogen bonds fromthe main chain atoms of the inhibitors, two from the P3 residue (theamide and the carbonyl groups) and one from the P1 residue (theamide group). However, the residues of the proteases that partici-pate in these hydrogen bonds come from different regions in theprimary sequences of the enzymes, due to the differences in theirbackbone folds (Fig. 6). In both chymotrypsin and subtilisin, thecatalytic serine residue is located spatially between the inhibitor (orsubstrate) and their hydrogen bonding partners in the protease. Theposition of the side chain hydroxyl of the catalytic serine would pre-vent the P1 carbonyl group from continuing the extended conforma-tion of the inhibitor/substrate. The main chain Ψ torsion angle ofthis residue is ~35°, compared to ~145° for the P4, P3, and P2

residues15,16. In this new conformation, the P1 carbonyl oxygen ispointed in the general direction of the oxyanion holes of the


enzymes. In addition, the hydroxyl of the catalytic serine is placedover the plane of this carbonyl group (Fig. 6). This arrangementtherefore very likely facilitates the attack of the serine hydroxyl on thecarbonyl carbon of the substrate and the subsequent stabilization ofthe resulting oxyanion. In HCMV protease, the inhibitor (substrate)is hydrogen-bonded to the β-strand in the protease that also containsthe catalytic serine residue. However, a twist in this strand also placesthe serine hydroxyl near the P1 carbonyl group (Fig. 6).

The oxyanion holes of the three enzymes (the amide nitrogen ofArg 165 in HCMV protease, the amide nitrogen of Gly 193 in chy-motrypsin, and the side chain amide nitrogen of Asn 155 in subtil-isin) are located within 1 Å of each other among the three enzymes(Fig. 6). The positions of the P1' residues of inhibitors of chy-motrypsin and subtilisin are rather similar as well (Fig. 6).Modeling a P1' residue based on the conformation of BILC 821showed that the P1' Ser residue can probably assume a similar con-formation when bound to HCMV protease. The S1' binding pock-et thus identified contains the strictly conserved Cys 161 residue(Table 2).

One feature unique to HCMV protease is the recognition of themain chain carbonyl of the P2 residue by the strictly conserved Arg165 side chain. This provides additional interactions between theinhibitor/substrate and the protease, and may be needed to com-pensate for the weaker interactions between the inhibitor/substrateside chains and the protease due to the requirement of a small P1

residue.

DiscussionThe residues of HCMV protease that show large conformationaldifferences between the inhibitor complex and the free enzyme arelocated mainly on the surface of the enzyme (with the exception ofthose in helix αF). In addition, these residues show reducedsequence conservation among the herpesvirus proteases2.Therefore, they are probably flexible and may be able to assumevarious conformations. This is confirmed by an inspection of theenzyme structure in the absence of substrate or inhibitors in fourdifferent crystallization conditions3–6. The structural differences we

Fig. 5 Comparison of the molecular surface31 near the active site of HCMV protease a, in complex with the inhibitor BILC 821, and b, in the freeenzyme structure3. The surface is colored based on electrostatic potentials. The inhibitor molecule is shown as a stick model (in green for carbonatoms). For reference, the inhibitor is also shown in (b). The overlap between the inhibitor P1 residue and the surface of the protease is due to thecovalent bond to the Ser 132 side chain. Large conformational differences in this region between the two structures produce a clear depression onthe surface for the S1 and S3 pockets in the inhibitor complex (a), whereas these pockets are not recognizable in the free enzyme state (b). One wallof the pockets, formed by residues Glu 31 and Arg 137, is absent in the free enzyme structure, as the residues are disordered or assume a differentconformation.

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observe between the free enzyme and the protease–inhibitor com-plex may be due to inhibitor binding and/or differences in crystal-lization conditions. However, as many of the residues that showstructural differences are located near the inhibitor, the pep-tidomimetic inhibitor probably plays an important role in stabiliz-ing their conformation. The change in fluorescence upon thebinding of peptidomimetic inhibitors suggests that the conforma-tion observed for the complex may only be sparsely populated inthe free enzyme state.

The activity of herpesvirus proteases is enhanced by glycerol,high salt and other kosmotropic agents18,19. In addition, the dimeris believed to be the enzymatically active species of this pro-tease20,21. The Kd of the dimer decreases from 8 µM in the absenceof glycerol to 2 nM in the presence of 25% glycerol21. The activityenhancement by kosmotropic agents is thought to be due mostlyto the stabilization of the dimer and the lowering of the Km. Theprotease–inhibitor complex shows that the P4 to P1' residues of theinhibitor do not directly interact with the other monomer of thedimer. However, the efficient hydrolysis of protein and peptidesubstrates requires more than the core recognition sequences ofthe P4 to P1' residues2. The smallest peptide substrates that can behydrolyzed by HCMV protease extend from the P4 to P4' positions.For herpes simplex virus protease, the smallest peptide substratesextend from the P5 to P8' positions. Based on the crystal structure,the other monomer could be involved in the binding of substrateresidues beyond the P3' positions. Further studies will be needed tocharacterize the structural impact of dimerization on substrate


recognition and catalysis. It is also possible that monomers ofHCMV protease possess some low catalytic activity.

Comparison of HCMV protease, chymotrypsin and subtilisindemonstrates the remarkable conservation of the serine proteasecatalytic machinery across different peptide backbone struc-tures22. HCMV protease thus provides another example of conver-gent evolution. The three hydrogen bonds from the P3 and P1

residues of the substrate to the protease help anchor the substrateto the active site. A deviation from the extended conformation isinduced at the P1 carbonyl group, which may be important forcatalysis. This is achieved by positioning the catalytic serineresidue between the substrate and its hydrogen bonding partnersin chymotrypsin and subtilisin. With HCMV protease, a newmechanism of inducing this change at the P1 carbonyl is observedwhich utilizes a twisting of the β-strand that contains both thehydrogen bonding partners and the catalytic serine residue.Additional mechanisms may be revealed from structural studies ofother clans of serine proteases and peptidases23.

The HCMV protease–inhibitor complex provides the basis forthe design of new inhibitors against the protease. Some of the fourhydrogen bonding interactions (Fig. 4) will probably need to bemaintained in the new inhibitors. The combined S1 and S3 bindingpockets, which appear to favor hydrophobic groups (Ala sidechain at P1 and Val side chain at P3), may be a good position forthese inhibitors to achieve favorable van der Waals interactionswith the protease. Establishment of stronger interactions in thisregion with the protease may also make it possible to remove the

Fig. 6 a, Conserved mode of bindingof peptide inhibitors (substrates) toHCMV protease (in cyan for carbonatoms), chymotrypsin (yellow), andsubtilisin (green). The catalytic triadsof the proteases and the P4 to P1'residues of the inhibitors are shown.The oxyanion holes are shown asspheres and colored similar to car-bon atoms. The greatest degree ofsimilarity is observed for the P2 andP1 residues. b, Conserved mode ofrecognition of the P4 to P1 residuesby (i) chymotrypsin, (ii) subtilisin,and (iii) HCMV protease. There is atwist in the β-strand near residue133 in HCMV protease, signified bythe dashed lines. The three con-served hydrogen bonds betweenthe inhibitor and the protease areshown as dotted lines. The sidechain hydroxyl of the catalytic serine

(with bold residue num-bers) is situated above theamide plane of the P1

residue, facilitating itsnucleophilic attack. Theposition of the serineresidue causes a ~90°change in the direction ofthe inhibitor backbone inchymotrypsin and subtilisin.

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activated carbonyl in the current series of inhibitors9. Furtherstructural, chemical and other studies will be needed to character-ize the detailed interactions to help the development of potentinhibitors of HCMV protease.

MethodsCrystallization and data collection. HCMV protease (A143Q,T181M and L229M triple mutant)3 was expressed in E. coli and puri-fied as described12. Purified protease was stored at 10 mg ml–1 (A280 =10) in 30 mM NaAc (pH 5.0), 80 mM NaCl and 1 mM DTT. The inhibitorBILC 821 was dissolved in DMSO to make a 200 mM stock solution. Forcrystallization, the protein stock solution was diluted to 7 mg ml–1

(~0.25 mM) concentration with buffer, and a small aliquot of theinhibitor stock solution was added to the protein to achieve four-foldmolar excess (or ~1 mM). Crystals were grown at room temperaturewith the hanging drop vapor diffusion method. The reservoir solutioncontained 18% PEG 4000, 0.1M HEPES (pH 7.5), 0.2M NaCl, 10% glyc-erol and 50 mM spermine. Typical crystals appeared as thin plates,measuring 0.3 × 0.8 × 0.03 mm3. The crystals were transferred in a fewsteps to an artificial mother liquor containing 30% PEG and 5% t-butanol, and then exposed to 0.15 M Na2SO4

3. An improvement inthe diffraction quality was also observed after the Na2SO4 treatment,although the ordered sulfate ion in the free enzyme structure3 is notpresent. An X-ray diffraction data set to 2.7 Å resolution was collect-ed at cryo-temperature on an R-Axis imaging plate system mountedon a Rigaku rotating anode generator (Table 1). Diffraction imageswere processed with DENZO24. The crystals belonged to space groupP21221 with a = 107.8 Å, b = 53.4 Å, and c = 212.4 Å. There are twodimers of the protease in the asymmetric unit. This unit cell is relatedto that of the free enzyme reported by us earlier3,11.

Structure determination and refinement. The initial structurewas determined by molecular replacement with the REPLACEsuite25, using the free enzyme dimer as the search model3. Rotationfunction and translation function solutions confirmed expectationsthat the dimers are located roughly in similar positions as those inthe free enzyme crystal3. The structure model was then subjected torigid body refinement using 5.0–3.6 Å reflections. This showed largemovements of the monomers relative to each other, suggesting adifferent dimer organization as compared to the free enzyme. Afterslow-cooling simulated-annealing crystal structure refinement withthe X-PLOR program26, a strong peak (~7σ) was identified in eachmonomer in the difference electron density map. The peak waslocated near the active site of the protease and was thereforebelieved to correspond to the iodine atom of the inhibitor.However, no electron density was visible for the rest of the inhibitorin the difference and the 2Fo – Fc maps.

Structure factors were calculated for all reflections to 2.7 Å reso-lution, using the atomic model after rigid body refinement and anoverall temperature factor value of 22 Å2. The calculated phaseswere applied to the observed reflections and the electron densitymap was solvent-flattened27, with a solvent content of 35%. The ori-entation and position of the two non-crystallographic two-fold axes


were optimized by electron density overlap with the REPLACE pack-age28. Subsequent four-fold NCS averaging among the two dimers,with a locally-written program (L.T., unpublished), produced a sig-nificant improvement in the quality of the electron density map.The inhibitor molecule was clearly visible in the electron densitymap. In addition, the map showed that there were large conforma-tional differences between this structure and that of the freeenzyme3, and that several loops missing in the free enzyme struc-ture had become more ordered. The atomic model from the freeenzyme was modified to fit the electron density map with the pro-gram FRODO29. Another cycle of solvent-flattening and four-foldNCS averaging was carried out starting with calculated phases fromthis new model (without the inhibitor). The inhibitor molecule wasthen built into the resulting electron density map (Fig. 1).

The structure refinement was carried out with the program X-PLOR26, for reflections between 6.0 and 2.7Å resolution withF>2σ. NCS restraints on the positions of main chain atoms were usedthroughout the entire refinement. Individual atomic temperaturefactors, tightly restrained by chemical connectivity and by NCS, wererefined after the completion of the positional refinement. No sol-vent molecules were included in the atomic model (Table 1). Tworesidues (Ser 54 and Ser 113) assume unfavorable main chain con-formations. Both are located in loops on the surface of the protein.

Coordinates. The atomic coordinates have been deposited in theBrookhaven Protein Data Bank (accession number 2wpo).

AcknowledgmentsWe thank W. Davidson for characterization of the protein samples by massspectrometry, C. Chabot for the synthesis of BILC 821, and D. Cameron for helpfulcomments.

Received 2 June 1998; accepted 20 July 1998.

Table 1 Summary of crystallographic information

Maximum resolution 2.7 ÅNo. of observations 275,527No. of unique reflections 33,502Rmerge 7.8%Resolution range for refinement 6.0–2.7 ÅNo. of reflections in refinement (F>2σ) 27,152Reflection data completeness 92%R-factor 22.6%RFree 33.2%R.m.s. deviation in bond lengths 0.011 ÅR.m.s. deviation in bond angles 1.6°No. of protein residues 920No. of inhibitor residues 20

Table 2 Substrate binding and inhibitor SAR of HCMV protease

Binding Site Residues Forming the Binding Site1 Residue Substrates Substrates Inhibitors3

(M-site)2 (R-site)2

S4 S134, S135, R136, R137, K156, P2 P4 L, T (V, A, P, I, H) Y des-amino Tyr, des-amino t-butylglycine, des-amino Val, acetyl

S3 E31, L32, S134, S135, R136, R137, R165, R166, P1 P3 V (L, I) L, V t-butyl, isopropyl, ethyl, adamantylS2 H63, S132, L133, S134, K156, H157, R165, P4 P2 N, D (Q, E) Q, K di-N-methyl Asn, Asn, Phe, His,

Gln,Leu, Lys, AspS1 L32, H63, S132, L133, G164, R165, R166, P3 P1 A A Methyl, EthylS1' 4 N62, H63, S132, A159, C161, V163 P1' S S (T, N) —

1Residues of HCMV protease located within 6 Å of the inhibitor are listed. Those residues that make major contributions to the binding pocket(hydrogen-bonding and/or van der Waals interactions) are highlighted in bold.2Based on a comparison of 13 herpesvirus proteases2. Residues that occur rarely (1 or 2 times) are shown in parenthesis.3Representative side chains with potencies within 10-fold of the best side chain (shown in bold) are given, in decreasing order of potency9.4These residues were identified based on a model for the P1' residue. See text for more details.


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Conserved mode of peptidomimetic inhibition and substrate recognition of human cytomegalovirus protease