Molecular Modeling Studies Suggest That ZincIons Inhibit HIV-1 Protease by Binding at CatalyticAspartatesDarrin M. York,12 Tom A. Darden, 1 Lee G. Pedersen, 2and Marshall W.Anderson1tLaboratory of Molecular Toxicology, National Institute of Environmental HealthSciences, Research Triangle Park, NC 27709 USA; 2Department of Chemistry, Universityof North Carolina at Chapel Hill, NC 27599-3290 USA

Human immunodeficiency virus type 1protease (HIV-1 PR) is a 99-amino acid,virally encoded protein responsible for pro-teolytic cleavage of viral gag and gag-polfusion polyproteins into functional prod-ucts (1-3). The activity of the protease isrequired for viral infectivity in vitro (4).Consequently, HIV-1 PR is an attractivetherapeutic target for rational drug design(5,6), and the focus of a tremendousamount of research.

HIV-1 PR has been characterized as anaspartic acid protease based on sequencehomology to related enzymes (7, catalyticpH studies (8), and inhibition by well-known aspartyl protease inhibitors (9,10).The active form of the enzyme behaves as adimer (11,12). These findings have beenconfirmed by the elucidation ofX-ray crys-tallographic structures of the enzyme bothunbound (13-15) and bound to syntheticinhibitors (16-20).

One feature that distinguishes theHIV-1 protease from cellular aspartyl pro-

teases is the pH dependence of its activity.The optimum pH range for activity of theHIV-1 protease (4.5-6.0) (8) is consider-ably higher than for cellular enzymes invitro (typically 2.0-4.0) (21). A notableexception is human renin, which has a pHoptimum in the range of 5.5-7.5 (22).The pH optima of these enzymes presum-ably reflect the catalytic mechanism gener-ally accepted for aspartyl proteases whichrequires one of the catalytic aspartateresidues to be protonated. Recently,Zhang et al. (23) reported that zinc ionsare inhibitors of both renin and HIV-1 PRat neutral pH. Although the zinc bindingsite(s) that lead to inactivation have notbeen determined, evidence suggests thatone site may occur at or near the catalyticaspartate residues. In this study, moleculardynamics methodology was used to explorethe possibility of zinc binding in the activesite of HIV-1 PR and predict the effects ofthis binding on the structure. Our resultsare consistent with the requirements ofzinc binding reported by Zhang et al. (23)and give supporting evidence that this isthe site that leads to inhibition of theprotease.

MethodsWe performed molecular mechanics anddynamics calculations using a modifiedversion ofAMBER3.0 (Revision A) (24,25).The all-atom force field (26) was used forall standard residues, and solvent was treat-ed explicitly using the TIP3P model (27).Parameters for chloride ions and zinc ionswere taken from Lybrand et al. (28) andBartolotti et al. (29), respectively. Elec-trostatic and van der Waals interactionswere treated using a "twin range" (9/18 A)residue-based cutoff, updated every 20steps. We performed simulations underconstant temperature (300 K) and pressure(1 bar) conditions using a 1-fsec integra-tion time step, carried out to 200 psec.

We obtained the starting positions ofthe heavy atoms for the unbound andZn2-bound dimers from the crystallo-graphic structure of the synthetic(Aba6795) HIV-1 protease at pH 7.0 (14).

The net charge of the unbound dimer wasassumed to be +4, consistent with the nor-mal protonation states of the componentamino acids at neutral pH. It is possiblethat the active site aspartates share a protonnear neutral pH, as maximum proteaseactivity occurs in the range 4.5-6.0 (8).However, inflections in the log V/K versuspH profile observed at pH 3.1 and 5.2suggest one of the aspartates is protonatedat pH 5.2. Since we were simulating theprotease at neutral pH, we chose to treatboth aspartates as being fully charged inthe simulations. In the case of the zinc-bound protease, it is reasonable to assumethat Zn2+ ion binding (pKa 9.5) (30)would involve both aspartates in theunprotonated form.

There was no particular bias in placing2+..a Zn ion in the active site a priori.

Instead, the goal was to find the positionof a Zn + ion resulting in the most favor-able ion-protein potential energy based onthe crystallographic coordinates for theprotein heavy atoms (with hydrogens ener-gy minimized by AMBER). Using thisposition for initial placement of the Zn2+ion, molecular dynamics may then be usedto refine the binding interactions anddetermine the resulting structural changesin the protein. Initial placement of a Zn2+ion was determined by a grid search proce-dure, whereby the AMBER nonbondinteraction energy (no cutoff) of the ionwas tested on a 0.5 A grid over a 5.0 A boxaround the crystallographic structure. Weused the grid points corresponding to the1000 lowest energies as starting points for. . . . r * ~2+.minimization of a single Zn ion to a rootmean square gradient tolerance of0.000001 kcal/mol. The position corre-sponding to the global potential energyminimum was chosen for the placement ofthe ion. Interestingly, this procedure

Address correspondence to D. M. York, NIEHS,PO Box 12233, Research Triangle Park, NC27709 USA. This work was funded in part by theUniversity of North Carolina Medical SchoolMD/PhD program, the National Cancer Institute,and the North Carolina Supercomputing Center.We thank A. Wlodawer for providing us with sev-eral crystallographic structures and for useful com-ments. We acknowledge grants of supercomputertime from the National Cancer Institute and theNorth Carolina Supercomputing Center. D.M.Y.thanks the University of North Carolina MedicalSchool for support through the MD/PhD pro-gram. L.G.P. thanks NIEHS for support for thisproject and acknowledges NIH grant HL27995.We also thank Howard Smith at NIEHS for essen-tial computer graphics assistance and NobukoHamaguchi and Paul Charifson for helpfulsuggestions.

Environmental Health Perspectives246

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resulted in the placement of the Zn2+ ionin the active site cleft directly between thecarboxylate side chains of the catalyticaspartate residues.

The net charge of the protease and pro-tease/ion complexes were neutralized withnegatively charged chloride ions using thesame procedure as for the zinc ion. Thesolute was solvated in a 13 A water bath(8013 water molecules) and equilibratedusing the same procedures as for previoussimulations (31). Unconstrained dynamicswas then performed on each system at con-stant pressure and temperature to 200psec.

ResultsFigure 1 shows the time evolution of theroot mean square deviation of a carbonatoms with respect to the crystallographicstructure for the molecular dynamics struc-tures of the unbound and Zn2+-boundprotease. Both systems are well equilibrat-ed after 150 psec. The unbound proteaseequilibrates with a carbon root meansquare deviation of about 1.5 A, whereasthe Zn2-bound structure equilibrates at alower value of approximately 1.2 A. Sec-ondary structural analysis of the crystallo-graphic structure and the simulation(150-200 psec) average structures was per-formed using the Kabsch and Sander pro-gram DSSP (32). Of the 63 secondarystructural assignments made for the crystal-lographic monomer, 46 (73%) were con-served in each monomer of the unboundstructure, and 54 (86%) were conserved ineach monomer of the Zn2+-boundstructure.

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X-ray Zn2+ bound UnboundContact Distance Distance % Time Distance % Time

atom 1-atom 2 (A) (A) bonded (A) bondedPro-1 HN+-Phe-99 0 1.71 1.79 100 1.73 100

1.76 100 2.85 46Pro-1 0-Phe-99 HN 2.16 2.13 86 2.07 93

lle-3 HN-Leu-97 0 1.68 2.12 88 2.09 882.12 88 2.03 93

lle-3 O-Leu-97 HN 1.78 1.97 97 1.97 991.96 100 1.97 99

Leu-5 O-Arg-87 HN21 2.16 1.97 96 2.03 871.91 98 3.39 28

Trp-6 O-Arg-87 HN22 2.23 66 2.60 162.09 88 3.81 13

Leu-24 0-Thr-26 HOG 2.55 1.90 99 2.05 931.88 100 1.91 100

Thr-26 HN-Thr-26 OG1 2.22 2.02 98 2.29 652.05 98 2.03 93

Gly-49 HN-Gly-51 0 2.06 94

Gly-51 HN-Gly-51 0 2.42 1.99 100

His-69 HNE-Phe-99 OXT 2.491.99 89

Thr-96 HN-Asn-98 0 2.41 2.03 93 2.00 982.03 95 2.01 99

Thr-96 O-Asn-98 HN 2.02 2.00 98 1.94 992.03 93 1.96 99

Hydrogen bonds at the dimer interface in the HIV-1 PR crystallographic structure and in the simulations.Hydrogens were added to the crystallographic structure (X-ray) using AMBER and energy minimizedkeeping the nonhydrogen positions fixed. In the unbound and Zn2+-bound simulations, the average H-bond distances and percent time the H bond was maintained [definition of an H bond as in York et al. (31)]are listed for each monomer (monomer 1 is listed on the same line as the H-bond atoms, monomer 2 islisted immediately below).

Hydrogen Bonding at the DimerInterfaceAn analysis of H-bond contacts at thedimer interface over the last 50 psec of thesimulations has been performed (Table 1).The primary hydrogen bond interactionsat the dimer interface (33) present in the

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Figure 1. Time evolution of the a carbon root mean square deviation from the crystallographic structureof Wlodawer et al. (14) for the unbound protease simulation (thick line) and the Zn2+-bound protease sim-ulation (thin line).

crystallographic structure are maintainedin solution both in the unbound and Zn2-bound simulations. These include interac-tions at the amino- and carboxy-terminal gstrands (residues 1-A, 95-99), which forma four-stranded antiparallel B sheet, and inthe region of the active site triads (residues25-27), which interlock in the "fireman'sgrip" characteristic of aspartyl proteases(33). In particular, inter-subunit hydrogenbonds involving residues at or near theactive site (Leu-24, Thr-26, and Arg-87)were all maintained in the presence ofbound zinc.

Zinc-Protein InteractionFigure 2 shows the pair distribution func-tion (34), gab(r), for the carboxylate oxy-gens of the catalytic aspartate residues (Fig.2a) and water oxygens (Fig. 2b) around theZn2+ ion and the corresponding runningintegration numbers, which indicate theaverage number of oxygens of each type ina sphere of radius r around the ion. Thepair distribution function for the carboxy-late oxygens shows a single, sharp peakcentered at 2.13 A, and the correspondingcoordination number, indicated by therunning integration number, is 4. Thepair distribution function for water oxy-gens shows there are three water moleculesassociated with the zinc ion in its first sol-

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r(A)Figure 2 Pair distribution function (34), gab(r), of oxygens around the zinc ion during the final 50 psec ofthe Zn2-bound simulation (thick lines) for (a) carboxylate oxygens of the catalytic aspartates and (b)water oxygens. The corresponding running integration numbers are also shown (thin lines).

vation sphere at a distance of around 2.25A. No exchange of oxygens in the firstcoordination sphere of zinc was observedduring the last 50 psec of the simulation.Zinc has been observed to be coordinatedto multiple carboxylate ligands in otherenzymes such as DNA polymerase I fromEscherichia coli (35).

Figure 3 is a stereo picture of theactive-site triads at the dimer interface withthe Zn ion bound to the catalytic aspar-tates. The crystallographic structure ofWlodawer et al. (14) is also shown super-imposed. Inter-subunit H bonds betweenThr-26-*Leu-24' and between Thr-26<-Thr-26' (primes indicate residues of thesymmetry-related monomer), termed the"fireman's grip," are clearly maintained.Additionally, the ODI atoms of the aspar-tate side chains maintain H-bond contactwith the backbone amino group of Gly-27on the same strand. These H bonds arealso pronounced in the crystallographicstructure.

The flap regions of the protease (res-idues 42-58), which are observed in crys-tallographic structures to significantlyrearrange upon inhibitor binding (16-20),were not observed to interact with the zincion in the simulation. Examination of the

Gly27'

Figure 3. Stereo picture of the active site triads (residues 25-27 and the corresponding symmetry related residues) for the 150-200 psec Zn2+-bound averagestructure showing zinc ion binding to the active site aspartates. The carbox,ylate oxygens of the catalytic aspartates are labeled "0," and inter-subunit hydrogenbonds are indicated by dotted lines [rz,2+0 = 2.13, 2.14 A (Asp-25); 2.10, 2.14 A (Asp-25')].

Environmental Health Perspectives248

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average structure of the zinc-bound pro-tease indicates the nearest backbone atomsof the flaps reside more than 9 A awayfrom the zinc ion.

DiscussionKinetic studies have shown that zinc is aneffective inhibitor of renin and HIV-1 PRat pH 7, two aspartyl proteases with pHoptima near neutrality (23). Other studiesof metal ion inhibition of HIV-1 PR per-formed at pH 5 did not reveal significantinhibition of the enzyme by Zn2+ ions(36). If inhibition of HIV-1 PR is a resultof Zn2+ binding at the active-site aspartateresidues, it is reasonable to expect that con-ditions that favor both aspartates in theunprotonated form would enhance Zn2+binding, and hence inhibition. Becauseboth catalytic aspartates presumably areunprotonated around neutrality, the ob-served pH dependence of Zn+2 inhibitionis consistent with binding at or near thecatalytic aspartates. Although the bacteri-ally expressed HIV-1 PR has three residuesthat commonly bind zinc (Cys-67, His-69,and Cys-95), Zn2+ also inhibits the relatedHIV-2 protease, which does not containcysteines or histidines [for a discussion ofHIV-2 protease, see Gustchina and Weber(37) and Tomasselli et al. (38)], in an iden-tical manner as the HIV-1 protease (23).This suggests that these residues are notrequired binding sites for inactivation byzinc. Considered with the fact that inhibi-tion was observed to be first order (23) inZn2+, we may infer that there is a singleimportant binding site that causes inactiva-tion. Furthermore, inactivation of the pro-tease by zinc was observed to be reversible,as protease activity could be restored uponaddition ofEDTA (23), which has a higheraffinity than carboxylate moieties for Zn2+ions. These data are, therefore, consistentwith the requirement of noncovalent bind-ing of Zn2 in the active site.

The present study of Zn2+ binding atthe catalytic aspartate residues provides evi-dence that this is the site responsible forinactivation of the protease. Evaluation ofthe potential energy of a Zn2+ ion in thefield of the crystallographic structure sug-gests a probable binding site for zincoccurs in the active site at the catalyticaspartates (see Methods). Subsequent mol-ecular dynamics simulations predict thatthe zinc ion remains stably bound in thisregion; i.e., no exchange of carboxylateoxygens was observed in the first coordina-tion sphere of the zinc ion after initialequilibration. Because the hydrolysismechanism mediated by HIV-1 PR re-quires one of the aspartates to be protonat-ed, forming a hydrogen bond with the car-bonyl oxygen of the scissile bond and theother in the unprotonated form to activate

a water molecule for hydrolysis (6), thecoordination of both of these residues by azinc ion would conceivably inactivate theenzyme. Gel filtration and equilibriumsedimentation analyses suggest that zincbinding does not involve disruption of thedimeric structure of the protease (23). Oursimulation with bound Zn2+ shows noweakening of the dimer. The overall simu-lation results indicate that Zn2+ can bindstably with the catalytic aspartate residuesof the HIV-1 protease without significant-ly disrupting the overall structure. Theseresults are consistent with experimentalstudies and taken together suggest that thesite of Zn + binding that leads to catalyticinhibition is the active site.

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