analysis of the structure of chemically synthesized hiv-1 protease complexed with a hexapeptide...

Analysis of the Structure of Chemically SynthesizedHIV-1 Protease Complexed With a HexapeptideInhibitor. Part I: Crystallographic Refinementof 2 Å DataMaria Miller,1*Maciej Geller,2,3 Michael Gribskov,1 and Stephen B. H. Kent41Macromolecular Structure Laboratory, NCI-Frederick Cancer Research Facility and Development Center, Frederick,Maryland2Laboratory of Mathematical Biology, NCI-Frederick Cancer Research Facility and Development Center, Frederick,Maryland3Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Warsaw, Poland4The Scripps Research Institute, La Jolla, California

ABSTRACT The structure of a complexbetween a hexapeptide-based inhibitor, MVT-101, and the chemically synthesized (Aba67,95,167,195; Aba: L-a-amino-n-butyric acid)protease from the human immunodeficiencyvirus (HIV-1), reported previously at 2.3 Å hasnow been refined to a crystallographic R factorof 15.4% at 2.0 Å resolution. Root mean squaredeviations from ideality are 0.18 Å for bondlengths and 2.4° for the angles. The inhibitorcan be fitted to the difference electron densitymap in two alternative orientations. Drasticdifferences are observed for positions and in-teractions at P3/S3 and P38/S38 subsites of thetwo orientations due to different crystallo-graphic environments. Proteins 27:184–194r 1997Wiley-Liss, Inc.†

Key words: aspartic protease; HIV-1; complexwith inhibitor

INTRODUCTION

The urgent need for developing anti-AIDS thera-peutics has prompted unprecedented progress inboth biochemical and structural studies on proteinsessential to the retroviral life cycle. Virally encodedaspartic protease (PR) was the first of the retroviralreplication enzymes for which the crystal structurehas been solved.1,2 The structure of the protease fromthe Rous sarcoma virus (RSV) was subsequentlyrefined at 2 Å resolution to an R factor of 14.8%.3 Thestructure of HIV-1 PR was independently deter-mined at 2.8 Å utilizing synthetic enzyme4 andsubsequently redetermined with recombinant en-zyme.5 These data have provided a unique opportu-nity for rational drug design, based on detailedknowledge of the three-dimensional structure of thetarget molecule. The design of competitive inhibitorswas facilitated by the advanced state of knowledge ofthe crystal structures of fungal and mammalian

aspartic proteases (Refs. 6 and 7 and referencestherein). Several sets of HIV PR inhibitors, whichwere developed based on the empirical knowledgefrom earlier renin inhibitors, also proved to beeffective in the case of HIV-1 PR.8 Even in thesecases, further structure-based investigations werenecessary to improve the potency and specificity ofdesigned PR inhibitors.From the structure of the unliganded enzyme it

was not possible to deduce the exact mode of sub-strate binding. Particularly intriguing was the roleof the flaps. Retroviral proteases are homodimericenzyme molecules, so two flaps could be involved inthe substrate binding. By contrast, the pepsinlikecell-encoded aspartic proteinases have only one flap.In the case of RSV PR crystal structure, the flapparts of the chains could not be traced in the electrondensity apparently due to their flexibility and conse-quent thermal motion within the crystal. In HIV-1PR the flaps were immobilized away from the activesite by contacts in the crystal lattice, and it waspossible to fit the peptide chain defining the flaps astwo extended b-hairpin structures.Solving the structure of the HIV PR molecule

complexed with inhibitors was necessary to deter-mine the interactions of the substrate with the flapsand the structural role of the solvent. The first suchstructure to be reported was the complex of chemi-cally synthesized HIV-1 PR9 with the reduced pep-tide bond hexapeptide (MVT-101).10 This revealedsubstantial changes in the protein backbone uponinhibitor binding.As expected, the largest movementinvolved flap regions on both monomers, where the

Contract grant sponsor: National Cancer Institute; contractgrant sponsor: DHHS, contract grant number NOI-CO-46000.Dr. Gribskov’s current address is San Diego Supercomputer

Center, P.O. Box 85608, San Diego, CA92186-9784.*Correspondence to: Dr.MariaMiller, Macromolecular Struc-

ture Laboratory, NCI-Frederick Cancer Research Facility andDevelopment Center, P.O. Box B, Frederick, MD 21702.Received 1August 1996; accepted 8August 1996.

PROTEINS: Structure, Function, and Genetics 27:184–194 (1997)

r1997WILEY-LISS,INC.†This article is aUSgovern-

ment work and, as such, is in the public domain in theUnited States of America.

change of positions for the tips of both flaps was asmuch as 7 Å. The coordinates from this 2.3 Åresolution structure have been used for a variety ofpurposes. The shape of the active site facilitated asearch of Cambridge Data Bank for nonpeptidecompounds that could potentially provide a good fitto the enzyme.11 The results of multiple copy simulta-neous search (MCSS)method applied to the construc-tion of peptide ligands in the binding site of HIV-1PR were first tested with the MVT-101 complexstructure.12 These coordinates were also used formodeling studies of inhibitor binding to HIV-2 prote-ase,13 and to determine the x-ray crystal structuresof two different peptide isostere inhibitors whichcocrystallized with synthetic PR in the same spacegroup,14,15 as well as the structures of several com-plexes with recombinant enzyme.16 The structurewas used to develop the techniques of moleculardynamics simulation in a crystalline environment17

and for molecular mechanics analysis of inhibitorbinding to HIV-1 protease.18Anumber of pharmaceu-tical companies have pursued x-ray crystal structureassisted design of PR inhibitors, and structural dataare now available for complexes in a variety ofcrystal forms (reviewed in Refs. 19–21). The newgeneration of potential anti-AIDS drugs aimed toinactivate PR, and consequently viral replication,has been successful in clinical trials and some com-pounds are now entering therapeutic use.22–24

It soon became clear that higher resolution struc-tural data were needed to provide a basis for theevaluation of the details of enzyme—substrate inter-actions, especially with the catalytic aspartates. Thestructure of a complex of HIV-1 PR with C2 symmet-ric inhibitor25 was reported at 2.8 Å resolution inhexagonal, instead of orthorhombic space group, anddid not assist in elucidating the effects of crystal con-tacts on the observed asymmetry of the complexes. Insome PR-inhibitor complexes, suspicious contacts be-tween the carboxylate oxygen of the active site aspar-tate and atoms of groups replacing the scissile bond(e.g., 2.6 Å to the carbon of the reduced peptide link incase of MVT-101 and 2.4 Å to oxygen from hydroxyl ofhydroxyethylene linkage in U-85548e15), made infer-ences with respect to the mechanism of enzymaticproteolysis ahighly speculativeundertaking. In the caseof a 2 Å structure of recombinant enzyme cocrystallizedwith acetylpepstatin,26 the inhibitor was reported asdisordered in the crystal lattice with essentially equaloccupancies of the two alternative orientations (esti-mated as 48% and 52%). This finding seems to shedsome light on the nature of observed distortions of thecontacts within the active site, since distances betweenstatine oxygen and aspartic carboxylate refined to 2.0 Åand 2.5 Å for the two alternative orientations. In thispaper we report the results of further refinement of theoriginal cocrystal structure of chemically synthesizedHIV-1 protease complexed with the reduced isosterehexapeptide inhibitorMVT-101 at 2Å resolution.

MATERIALS AND METHODS

HIV-1 PR used in this study was prepared by totalchemical synthesis.4,9 Its sequence corresponds tothe SF2 isolate of HIV where cysteines 67 and 95 ineach subunit were replaced by L-a-amino-n-butyricacid. The hexapeptide inhibitor MVT-101 had thesequence:N-acetyl-Thr-Ile-Nle-c [CH2-NH]-Nle-Gln-Arg · amide and was prepared by chemical synthe-sis. This inhibitor was designed as an analog of theCA/NC cleavage site of the viral gag-pol polypro-tein,27 in which the scissile peptide bond has beenreplaced by a reduced peptide bond. Crystals of thecomplex were obtained by cocrystallization as previ-ously described.10 The space group is P212121 witha 5 51.7 Å, b 5 59.2 Å, c 5 62.45 Å and the asym-metric unit contains a protease dimer with one boundinhibitor molecule. X-ray diffraction data extendingto 2.0 Å resolution were collected using a Siemenselectronic area detector from two crystals and wereprocessed, combined and scaled using the XENGENpackage.28 A total of 99662 observations (I $ 1.5s)were reduced to 12,163 unique reflections out of13,282 possible to 2 Å resolution; the merging R-symon intensities was 0.095. For the last shell (2.12 Åto 2.0 Å) the data were 75% complete. The results ofthe measurement are summarized in Table I.

Refinement of the 2 Å Data

The model reported previously10 was further re-fined utilizing data in 10 to 2 Å resolution range, us-ing the restrained least square refinement programPROLSQ,29 in a version which implemented fastFourier transform, PROFFT.30 Themodel wasmanu-ally rebuilt utilizing the program FRODO31 on anEvans and Sutherland PS390 graphics system. Pro-gram X-PLOR32 version 2.1 was used to calculateseveral simulated annealing omit maps. Compari-sons and alignments were performed with the pro-gramALIGN.33

The starting model was characterized by an Rfactor of 0.176 at 2.3 Å resolution. This model

TABLE I. Statistics for theData Set

Shell lowerlimit (Å)

Number of reflections

Rmerge*Possible Collected Observed

3.63 2343 2343 26636 0.0592.88 2220 2220 23463 0.1042.52 2192 2179 21296 0.1692.29 2203 2037 19736 0.2332.13 2155 1765 8154 0.2652.00 2169 1619 6377 0.334Total 13282 12163 99662 0.962

*Rmerge 5 S(Ii j 2 Gi j 7I 8j)/S 0 Ii j 0

where

Gi j 5 gi 1 Aisj 1 Bisj2; s 5 sin u/l

g, A and B are scaling parameters.

185HIV-1 PR/MVT-101 COMPLEX

included 70 water molecules numbered 501–570. Incorrespondence with the coordinates deposited withthe Protein Data Bank (set 4HVP), the numbers 1 to99 were used to designate amino acid residues of thesame monomer in the crystal lattice,10 residues ofthe second monomer have their corresponding num-bers increased by 100 (i.e., 101 to 199) and thereduced hexapeptide inhibitor residues are num-bered 201 to 206.Higher resolution data were included in a single

step. After three rounds of PROFFT refinement alter-nating with manual intervention sessions, 30 newwater molecules were added to the model, and the Rfactor improved to 0.164. In spite of several attemptsto rebuild the inhibitor backbone, after refinementthe carbon atom of the reduced peptide link persis-tently made a too short contact (2.6 Å) with thecarboxylate oxygen of one of the catalytic aspartate

side chains and proper geometry of the link wasnever retained (see Table II). Also, although thequality of the electron density map was very good forthe peptide chains for most of each subunit, at thisstage of the refinement additional features becamevisible aroundGln-205 of the inhibitor. Together theseinconsistencies suggested the possibility of staticbidirectional binding of the inhibitor in the crystallattice. These featureswere evenmore pronounced ona difference Fourier map calculated after 15 cycles ofPROFFT with the inhibitor removed from phasing.The hexapeptide inhibitor oriented in the opposite

direction was then fitted de novo to this map. In thenew orientation, five residues of the hexapeptide(residues 201–205, excluding Arg-206) seemed to fitthe electron densitymore closely. In this new orienta-tion, the geometry within the active site, both bondangles and distances, were acceptable after the

Fig. 1. a: Side chains ofArg-8. b: Side chains ofAsp-25. Modeled as disordered, correlating withthe static disorder of the inhibitor. 2 0Fo 0 2 0Fc 0 electron density map in this region is contoured at the1.0s level.

TABLE II. Geometry of VariousModels of theActive Site of theMVT101–PRComplexAfter Least-SquaresRefinement

Refinement with the inhibitorpresent in one orientation

Refinement with the inhibitor presentin two alternative orientations

occA 5 1 occB 5 1 occA 5 0.5; occB 5 0.5 occA 5 0.3; occB 5 0.7

d(C*, Od225,125) 2.6 3.0 2.7 3.2 2.8 3.1d(N*, Od225,125) 3.3 2.9 3.4 2.9 3.2 2.3d(Od125, Od1125) 2.6 2.6 2.5 2.7 2.6 2.7a(C*) 116.4 111.7 104.9 104.1 104.6 107.2a(N*) 119.3 123.2 116.9 119.0 117.6 120.67Biso8inhibitor 18.1 18.0 14.6 7.2 10.8 11.2R factor 16.1 15.9 15.5 15.4

d, close contacts (Å); a, bond angle (degree); 25,125, catalytic aspartates; occ, occupancy.*Atoms of the reduced peptide link.

186 M. MILLER ET AL.

refinement (see Table II). In the initial model of thecomplex,10 the guanidinium side chain of the C-terminal arginine (Arg-206) of the inhibitor dis-placed the Arg-8 side chain from the first monomer,taking its place in an ionic interaction with Asp-129.34 No such displacement was detectable forArg-108, the corresponding side chain in the othersubunit, so the side chain guanidinium group ofArg-206 in the new orientation (orientation B) wasplaced in the density corresponding to a watermolecule in the previous model of the complex.

Neither of the orientations of the hexapeptideinhibitor could account for the whole electron den-sity, and it became clear that both orientations mustbe present in the crystal lattice. Static disorder wasmodeled for Arg-8 (Fig. 1a) and two water moleculeswere added with partial occupancies. The same typeof static disorder had been reported for severalcomplexes of the HIV-I protease with peptide inhibi-tors in orthorhombic26,35 and hexagonal36 spacegroups. Such static disorder should not be surprisingfor a symmetrical dimer.

Fig. 2. Stereo view of the two alternative orientations ofMVT-101 hexapeptide. a: Initial 0Fo 0 2 0Fc 0 electron density mapcontoured at the 1.25s level based on the preliminary refinement(10–3 Å; data from the first crystal10) of the free enzyme model. b:

2 0Fo 0 2 0Fc 0 electron density map (contoured at the 1.0s level)calculated after the final refinement. Red line corresponds to theorientation B (70% occupancy) and the green line to the orienta-tion A (30% occupancy).


In an attempt to reduce the ambiguities in fittingthe two orientations of the hexapeptide into overlap-ping density, each orientation was first refined sepa-rately with 50% occupancy. The difference Fouriermap with ( 0Fo 0 2 0Fc 0 ) coefficients was used as aguide for corrections in the fitting of the inhibitor inthe opposite direction. Subsequently, each orienta-tion was correlated with the corresponding positionof Arg-8, and static disorder refinement was carriedout utilizing this option of PROLSQ, initially withoccupancies for both orientations set at 50%. Thisresulted in a mean thermal factor of 14.6 Å2 fororientation A and 7.2 Å2 for orientation B. Featuresof the model at different stages of refinement aresummarized in Table II.The observed geometry of the reduced peptide link

of orientation A improved significantly but the con-tact of the reduced peptide bond CH2 carbon with Od2

from Asp-25 was still an unusually short 2.7 Å. Thiscould be improved only by modeling both catalyti-cally active aspartates as disordered as well. Suchapproach seemed to be justified by the appearance ofthe electron density in this region (Fig. 1b), whichshowed an overlarge region corresponding to theAsp-125 side chain. Side chains for both aspartateswere fitted separately for both orientations of theinhibitor. After regularization within FRODO it be-came necessary to model disorder for the triads ofresidues surrounding the catalyticAsp residues (resi-dues 24–26 and 124–126). Using the same approachas described by Fitzgerald et al.,26 occupancies forboth orientations were adjusted until their meanthermal factors became equal. This procedure re-sulted in assigning occupancy of 30% and 70% for theorientations A and B, respectively. Unmerged struc-ture factors from each crystal were than checked instatic disorder refinement with similar results, thatis, orientation B was the dominant one in both cases.Merged data from two crystals were used for furtherrefinement to ensure suficient redundancy and com-pleteness of the observations. The superposition ofthe two alternative orientations of the inhibitor andthe initial electron density map is shown in Figure2a and the map calculated after the static disorderrefinement in Figure 2b. Refinement statistics of thefinalmodel are listed in Table III, and the Ramachan-dran plot for the protein part of the complex is shownin Figure 3.Most of the main chain of the enzyme is very well

defined by the electron density. The poorest densitywas observed for residues 14–18 in each mono Thisregion constitutes a b turn on the surfacemer. of themolecule, moreover in this space group it is in closecontact with corresponding region (residues 114–118) of symmetry-related molecule, causing disorder.Both turns were modeled as type II b hairpins as forthe structure of unliganded enzyme. A simulatedannealing omit map37 calculated for this region didnot show any new features at this stage of refine-ment, but confirmed the accuracy of the model.

RESULTS AND DISCUSSIONDescription of the Crystal Structure

Themodel of the HIV-PRmolecule complexed withMVT-101 derived from the 2 Å diffraction dataconsists of two crystallographically independent sub-units, 116 water molecules and a hexapeptide inhibi-tor. In about 70% of the molecules in the crystallattice, N-terminal of the inhibitor binds to monomer1, while for the remaining 30%, the bound inhibitor

Fig. 3. Ramachandran plot for the protein part of the complex.Glycines are marked by (o), all other amino acid residues by (1).

TABLE III. Final Statistics of theMVT-101PRComplex

R factor 5 S\Fo 0 2 0Fc\/S 0Fo 0 0.154Resolution 10.0–2.0 ÅNo. of reflections 10,657No. of atoms 1,762rms deviations from ideality*Distance restraintsBond distance 0.018 (0.020) ÅAngle distance 0.053 (0.040) ÅPlanar 1–4 distance 0.057 (0.050) Å

Plane restraints 0.014 (0.018) ÅChiral center restraints 0.166 (0.150) Å3

Nonbonded restraintsSingle-torsion contact 0.197 (0.300) ÅMultiple-torsion contact 0.206 (0.300) ÅPossible (H···Y) H bond 0.207 (0.300) Å

Conformational torsion anglesPlanar 2.7 (3.0) °Staggered (19.1) (10.0)°Orthonormal 10.8 (20.0) °

Biso restraintsMain-chain bond 1.457 (1.500) Å2

Main-chain angle 2.182 (2.000) Å2

Side-chain bond 4.386 (3.000) Å2

Side-chain angle 6.333 (4.000) Å2

H bond 12.534 (15.000) Å2

*Target restraints in parentheses.


has the opposite orientation. The hexapeptide inhibi-tor residues are numbered 201 to 206 with the letterA or B to differentiate between the two possibleorientations. Orientation A corresponds to the onedescribed previously,10 but is the minor one in thepresent model. Some residues in the enzyme mol-ecule were also modeled in two conformations withoccupancies that correlate with the observed staticdisorder of the inhibitor; those were the side chain ofArg-8 and the atoms of the residues 24–26 and124–126. A 2 0Fo 0 2 0Fc 0 map in the vicinity of Arg-8and Asp-25 after final refinement is shown in Figure1. Water molecules 414 and 415 had an assignatedoccupancy of 70%. Wat-511 from the previous modelhas been renumbered as Wat-301, following theusage from other papers on this subject.14,15

The overall view of the complex is shown in Figure4. The main chain of the inhibitor lies at a skewedangle with respect to the long axis of the dimericenzyme molecule, in a position that allows optimalinteractions with the flaps (residues 45–55 and145–155) from both monomers. Planes of the b-hair-pin loops of the flaps are positioned in such a wayrelative to the inhibitor backbone that only thebottom edge of each loop (residues 46 to 50 and 146 to150) interacts with the inhibitor. Both ends of theinhibitor are in contact with the environment which

in this case is not crystallographically equivalent.The two subunits of PR-dimer are related by a pseudo-twofold axis of symmetry (perpendicular to the planeof Fig. 4), which deviates from ideality by 1.5° (forsuperposition of corresponding Ca atoms of bothmonomers). This is even less than in the case of theunliganded protease fromRous sarcoma virus, whichalso crystallized as a dimer in the asymmetric unit.3

The distribution of the differences in positions forCa from both subunits is shown in Figure 5c. Threeregions with the largest deviations from perfectsymmetry (residues 14–18, 37–39, and 79–81) be-long to the surface loops,4 which are most prone todistortions due to crystal packing forces. The differ-ences between the two subunits can be easily ex-plained by different crystal contacts. The residuesinvolved in crystal contacts are listed in Table IV.Loops 14–18 and 37–39 interact with correspondingparts from symmetry-related second monomer. Ofparticular interest are loops 78–82 from both mono-mers since they constitute the walls of the active sitecavity. This part of monomer 1 make extensivecontacts with the molecule related by symmetryoperation2x, 1⁄2 1 y, 1⁄2 2 z. Trp-106 of the symmetry-related molecule (#Trp-106) stacks over Pro-79 sothat the planes of the two rings are only 3.7 Å apart.The Pro-79 ring is pushed toward the interior of the

Fig. 4. The stereo view of the Ca tracing of the protease dimer with the bound hexapeptideshown in the ball and stick representation.


dimer, making the active site cleft slightly asymmet-ric. Moreover, the a-carbonyl of Pro-81 forms ionicinteraction with the side chain amide of #Gln-107,and Cb of Pro-79 is 3.6 Å from Cg2 of #Thr-104, leav-ing little space for flexibility. The same #Trp-106 is inclose contact with the a-carbonyl oxygen of Gly-149

and Ce1 Phe-153, and the flap of the second monomerlies closer to the symmetry axis of the dimer.For comparison, the same type of a diagram is

shown in Figure 5b for the PR-complex with theheptapeptide-derived hydroxylethylamine inhibitorJG-365,14 which was crystallized in the same spacegroup. JG-365 is a subnanomolar inhibitor and wasreported to bind predominantly in the orientationopposite to the major orientation of MVT-101, that isin the same direction as the MVT-101 orientation A.The strikingly similar pattern for the complexes withthe two inhibitors is consistent with the observeddeviation from twofold symmetry arising from thedifferent crystallographic environment of the twosubunits, rather than from the bound inhibitors.The tips of the flaps are situated very close to each

other and as a result their conformation is restricted.In order to avoid unfavorable contacts between thea-carbonyl oxygens of residues 50 and 150, only typeII b-turns would be possible for the fully symmetri-cal dimer. In this crystal lattice the peptide bondbetween residues 50 and 51 is turned approximately180° in respect to the other monomer and a chain ofcooperative hydrogen bonds is formed: O79···Wat-305···N151, O150···N51, O50···Wat-345. The samearrangement was observed for the complex withJG-36514 in the same space group, which was foundto bind to the enzyme predominantly in the oppositedirection than major (orientation B) of MVT-101.Neither oxygen 50 nor 51 is in close contact with anyatom from the inhibitor, thus no correlation betweenconformation of this amide and the orientation of theinhibitor can be found. In solution, the conformationof both flaps may be equivalent and a mixture of flapconformers may be formed.For a similar reason a second break from the true

symmetry occurs at the dimer interface formed by

Fig. 5. The rms deviations between corresponding atoms of the two enzyme subunits after theleast-squares alignment of their main-chain atoms. a: U-85548e PR complex.15 b: JG-365 PRcomplex.14 c: MVT-101 PR complex.

TABLE IV. Crystal Contact: Pairs of ResiduesAreListed forWhichAtomsAre in Shorter Contacts

Than 3.8 Å

Arg-14 Arg-114Gly-16 Pro-139Gln-18 Pro-139Met-36 Asn-137Asn-37 Pro-139Leu-38 Asn-137Pro-39 Asn-137Pro-39 Gly-116Gln-61 Pro-144Gln-61 Lys-145Leu-63 Gly-117Pro-79 Thr-104Pro-79 Trp-106Pro-81 Trp-106Pro-81 Gln-107Thr-91 Phe-153Gln-92 Phe-153Gln-92 Met-146Trp-106 Gly-149Trp-106 Phe-153Thr-112 Lys-141Leu-119 Trp-142Gln-2 Glu-135Trp-6 Pro-181Leu-19 Pro-44Trp-42 Gly-67Phe-53 Gln-192Phe-53 Lys-170


intertwining N and C termini of both monomers. Toavoid unfavorable contacts between the side chainoxygen and nitrogen of Asn-98 and Asn-198, theplanes of their carboxy-amides are positioned almostperpendicular to each other. This facilitates the for-mation of a string of cooperative electrostatic interac-tions across the b sheet: Oe1(Gln-2)···Nd2(Asn-198),Od1(Asn-198)···Nd2(Asn-98), Od1(Asn-98)···Ne2(Gln-102)(see also Ref. 34). This asymmetry is further main-tained due to the intrasubunit hydrogen bonds involv-ing Thr-96 and Thr-196. Within the first monomerOg1 from Thr-96 is an acceptor of a proton fromNd2 ofAsn-98, while within the second monomer, Og1 ofThr-196 donates the proton to the hydrogen bondwithOd1 from Asn-198. This dimer interface region hasbeen modeled as disordered in the structure of HIV-1PR complexed with acetyl-pepstatin,26 which crystal-lized in space group P21212. In ourmodel this networkwas refined in one orientation, and Ne2 from Gln-2forms a short hydrogen bond with the side chain car-boxylate of Glu-135 from symmetry-relatedmolecule.

Interactions of the Inhibitor With the Enzyme

The backbone dihedral angles for the alternativeorientations of MVT-101 are compared in Table V.Four central residues from both orientations arerelated by the same noncrystallographic twofoldsymmetry as the subunits of the dimer. Torsion

angles for P3 and P38 sites inAand B orientation aredramatically different. The dominant (i.e., B) orienta-tion will be described in detail, and then comparedwith the opposite one.Positioning of the inhibitor in orientation B in the

active site cleft is shown in Figure 6 and the poten-tial hydrogen bonds in the case of both alternativeorientations are listed in Table VI. The hexapeptideis in an extended conformation, but its backbone isslightly bent, so thatmain chain–main chain interac-tions are formed with both the flaps and the body ofthe enzyme. Six direct hydrogen bonds are formedbetweenmain chain carbonyls andNH groups, whichinclude Gly-27, Asp-29 and the carbonyl of Gly-48.These interactionswith the PRmonomers are equiva-lent for both orientations of the inhibitor and havebeen seen in all structures of peptide-based inhibitorcomplexes reported so far.20,21

Hydrogen-bonding interactions between nitrogens50 and 150 from the flaps and inhibitor carbonyls oneither side of the scissile bond are mediated by aburied water molecule (Wat-301). The characteristicfeature of HIV-1 PR complexed with substrate-derived inhibitors was first observed in the original2.3 Å structure10 and has also been seen in othercomplexes.19–21 The amide NH from Gly-48 partici-pates in a hydrogen bond with a carbonyl oxygen ofbound peptide, either the oxygen of N-terminalacetyl or the a-carbonyl of the C-terminal Arg for Aand B orientations of MVT-101, respectively. Theamide NH of Gly-148 is 4.48 Å from Og1 of Thr-201-Bor 3.6 Å from carbonyl oxygen of Arg-206A. Nochange in relative orientation of the two subunits ofthe dimer is necessary to maintain these interac-tions with the backbone of the bound peptide, whichbinds in a pseudosymmetrical way. On the otherhand in comparison with the structure of the unli-ganded enzyme, (c, f) pair of dihedral angles ofGly-27 changed from (272°, 282°) to (292°, 15°) and(2102°, 19°) in subunit 1 and 2, respectively, in thecase of the complex. As a result, the carbonyls of

Fig. 6. Stereo view of the hexapeptide (solid line) in the active site. Hydrogen bonds are shownas dashed lines.

TABLEV. TheBackboneDihedralAngles (Degrees)forAandBOrientations of the Inhibitor

Residue

f c v

A B A B A B

Thr-201 132 29 2177 180Ile-202 2117 2114 96 86 179 176Nle-203 294 297 69* 57* 135* 148*Nle-204 277* 264* 154 147 180 2177Gln-205 2159 2153 97 134 2178 180Arg-206 274 2125 22 2125

*Reduced peptide link.


Gly-27 and Gly-127 turned approximately 90° to-ward the inhibitor, allowing formation of hydrogenbonds with amide NHs from P28 and P1 sites. Also,planes of the carboxyl groups of Asp-29 and Asp-129are rotated about 90° relatively to their positions inthe free enzyme, allowing Od2 from Asp-29 partici-pate in a hydrogen bond with nitrogen of N-terminalamide of bound MVT-101. This change is symmetri-cal on the second subunit, although the side chain ofAsp-129 does not interact directly with this inhibitorin the major orientation. It seems that this rotationis caused by the proximity of a-carbonyls from P3and P28 sites.Positioning of the reduced peptide link with re-

spect to the active site aspartates is very similar to

that described for the complex of reduced peptideinhibitor with PR from Rhizopus chinensis.38 In caseof orientation B of MVT-101 with the HIV-1 PR, thecarbon of the reduced peptide bond is positioned 3.2Å from Od2(25) and 3.5 Å from Od2(125), while theamino nitrogen is in hydrogen bond distance (3.0 Å)from Od2(25). As seen in Table II, the distances fororientation A are still distorted. This is due tounresolvable superposition of x-rays scattering fromboth orientations of the inhibitor present in onecrystal and it is consistent with assigning loweroccupancy for this orientation.Contacts between the side chains of the inhibitor

and protein residues (listed in Table VII) define sixor seven specific binding pockets along the active sitecleft. Only the four central ones are well defined andthey do not include solvent molecules, other thanWat-301.

S1/S18 PocketAlthough norleucine sites comprise both the P1

and P18 sites on the inhibitor, the two correspondingpockets are quite asymmetric. Due to distortioncaused by crystal packing forces (see above), S1pocket is more compact and it includes the polar sidechain of Thr-80, with its Og1 pointing toward interiorof the subsite. A glutamate side chain can be easilymodeled in the place of norleucine in such a way thatits carboxylate oxygens would accept protons fromthe hydroxyl of Thr-80 and the amide nitrogen ofIle-50. The side chains of Ile-50 and Ile-150 areturned approximately 90° relative to each other.Similar side chain asymmetry is observed for thepair Ile-84, and Ile-184. Conformation of Ile-84 seemsto be correlated with the conformation of Ile-150 inthe same pocket and Ile-184 with Ile-50, apparentlyto avoid unfavorable contacts.

S2/S28 PocketInteractions between the P2 and P28 side chains

and the enzyme in the case of both MVT-101 orienta-tions are very similar. Except from Ile-50 and 150,only residues from onemonomer contribute to a givenpocket. In addition to nonbonded contacts betweenthe atoms of Ile-202 or Gln-205 and the enzyme, theside chain carbonyl oxygen of Gln-205B is an accep-tor in two hydrogen bonds with the main chain NH’sofAsp-29 andAsp-30, while its side chain amide formsa hydrogen bond with the carboxylate of Asp-30.

S3/S38 PocketsThese subsites include several residues from the

surface of the PR dimer. Inhibitor side chains fromP3 and P38 sites can also interact with solvent andsymmetry-relatedmolecules in the crystal. The tip ofArg-206B is positioned between Phe-53 and Pro-181,thus it is in a hydrophobic pocket, while the protonfrom the guanidinium group points toward the a-car-bonyl of Gly-9, providing additional favorable electro-static interactions. On the other side of the dimer,the N-terminal acetyl group occupies this subsite,and its methyl group is shielded from water by

TABLEVI. Potential HydrogenBond InteractionsBetween the Inhibitor and Its Environment

in theActive Site*

Enzyme/solvent

Distance fornonhydrogenatoms (Å)

Inhibitor orientationAOTacetyl N 48 2.98N 201 Od2 29 3.10O 201 N 29 3.30N 202 O 48 2.48O 202 O 301 2.27N 203 O 27 2.98N 204 Od2 25 3.19O 204 O 301 2.50N 205 O 127 3.09Ne2 205 Od2 130 4.13Oe1 205 N 130 3.27Oe1 205 N 129 3.76O 205 N 129 3.05N 206 O 148 3.60NH2 206 Od1 129 2.47O 206 O 397 2.81NTamide Od2 129 2.81

Inhibitor orientation BOTacetyl O 414 2.87N 201 O 148 3.02O 201 N 129 3.14O 201 O 415 3.20N 202 O 148 3.08O 202 O 301 2.65N 203 O 127 2.82N 204 Od2 125 2.96O 204 O 301 3.02N 205 O 27 2.90Ne2 205 Od2 30 2.78Oe1 205 N 30 2.90Oe1 205 N 29 2.96O 205 N 29 2.96O 205 O 304 2.90N 206 O 48 2.90NH1 206 O 49 3.71O 206 N 48 2.87NTamide Od2 29 3.08

*Probability of the formation of hydrogen bonds utilizingtwo-angle criterion during 100 ps of MD simulation is given inpart II of the paper.


#Trp-106 inserted between Pro-81 and Phe-153. Thr-201B is partially exposed to solvent, Wat-397 medi-ates interaction of hydroxyl 201B with carboxylatesof Asp-129 and Asp-130. In the molecules whereinhibitor is bound in the opposite orientation, proxim-ity of #Trp-106 forces the side chain of Arg-206A toadopt strained conformation. This steric hindrancein the crystal lattice may be the reason that orienta-tionAwas less populated in both analyzed cocrystals.

Important Water Molecules

Beside the well-characterized water 30110,19–21

there are four water molecules: Wat-302, Wat-304,Wat-307, andWat-415 which are important formain-taining the structure39 of the protease-inhibitor com-plex. These molecules are located symmetrically inhydrophobic pockets on each side of the active site:Wat-304 and Wat-307 between residues Thr-26, Gly-27, Asp-29, and Arg-87; Wat-415 and Wat-302 be-tween residues Thr-126, Gly-127, Asp-129, and Arg-187. Wat-415 is located 3.2 Å from the carbonyloxygen Thr-201 of the inhibitor, andWat-304 is 2.9 Åfrom the carbonyl oxygen Gln-205.

CONCLUSIONS

The results reported here indicate that great careis requiredwhile interpreting data from other cocrys-tals of inhibitors with the HIV-1 PR, especially thoseextending only to low or medium resolution. Refine-ment of 2 Å data indicated bidirectionality of theinhibitor in the crystal lattice. Modeling of MVT-101in two alternative orientations allowed for correc-tions of several inaccuracies in the original 2.3 Åmodel. The 2 Å structure describes much moreaccurately the juxtaposition of the reduced peptidelink of the inhibitor and the catalytic aspartates aswell as the interactions between the P2 and P28 sidechains and the enzyme. This is in agreement withMCSS calculations done with MVT-101 sequence,which showed that the P2-P28 segment of the com-puted structure characterized by the lowest totalenergy is also the closest (all atom rms deviation of1.76 Å) to the corresponding segment of theMVT-101in its major orientation described here.12

No asymmetry between the two subunits of theprotease dimer which could be attributed to thedirection of the inhibitor binding could be detected at

this resolution. All the intersubunit differences arelocated on the surface of the molecule and can beexplained by interactions with the different crystallo-graphic environments. On the other hand, the com-parison of the two alternative conformers of MVT-101 clearly shows that the conformation of both endsof the bound hexapeptide depends on the environ-ment of the complex. Thus the specificity of P3/P38

sites cannot be unambigously determined from thecrystal structure alone. Molecular dynamic simula-tion in water solvent should help in elucidating theinfluence of crystal contacts (see accompanying pa-per).The data described here rationalize further our

understanding of the substrate/inhibitor bindingand specificity and should help in structure-baseddesign of inhibitors for the HIV-1 protease, an impor-tant target for the development of anti-AIDS thera-peutics.

ACKNOWLEDGMENTS

We are grateful to Dr. Alexander Wlodawer for hishelp and support. This research was sponsored inpart by the National Cancer Institute, DHHS, undercontract no. N01-CO-46000 withABL. M. Geller wassupported in part by grant KBN-4.0078.91.01 (StateCommittee for Research, Poland). The contents ofthis publication do not necessarily reflect the viewsor policies of the Department of Health and HumanServices, nor does mention of trade names, commer-cial products, or organizations imply endorsementby the U.S. government.

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TABLEVII. Enzyme-BindingPockets forMVT-101 inMajorOrientation

P4Acetyl P3 Thr P2 Ile P1 Nle P81 Nle P82 Gln P83Arg

Pro-81 Arg-8 Ile-50 Asp-25 Asp-125 Ile-150 Arg-108Gly-148 Gly-148 Ala-128 Thr-80 Ala-28 Gly-48Wat-414 Asp-129 Val-132 Pro-81 Pro-181 Val-32 Ala-28Wat-312 Ile-147 Ile-147 Val-82 Val-182 Ile-47 Met-46

Wat-312 Gly-148 Ile-84 Ile-184 Gly-48 Gly-49Wat-397 Ile-184 Gly-127 Gly-27 Ile-84 Phe-53Wat-408 Gly-149 Asp-29 Pro-181

#Tryp-106 Ile-150 Ile-50 Asp-30Leu-23 Leu-123

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analysis of the structure of chemically synthesized hiv-1 protease complexed with a hexapeptide...

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