Quantum Computational Analysis for Drug Resistanceof HIV-1 Reverse Transcriptase to Nevirapine ThroughPoint MutationsXiao He,1 Ye Mei,1 Yun Xiang,2 Da W. Zhang,2 and John Z.H. Zhang1,2*1Department of Chemistry, Institute of Theoretical and Computational Chemistry, Key Laboratory of Mesoscopic Chemistry ofMinistry of Education (MOE), Nanjing University, Nanjing 210093, People’s Republic of China2Department of Chemistry, New York University, New York, New York

ABSTRACT Quantum chemical calculation hasbeen carried out to analyze binding interactions ofnevirapine to HIV-1 reverse transcriptase (RT) andsingle point mutants Lys103 3 Asn (K103N) andTyr1813 Cys (Y181C). In this study, the entire sys-tem of HIV-1 RT/nevirapine complex with over 15,000atoms is explicitly treated by using a recently devel-oped MFCC (molecular fractionation with conju-gate caps) approach. Quantum calculation of pro-tein–drug interaction energy is performed atHartree-Fock and DFT levels. The RT-nevirapineinteraction energies are computed at fixed geom-etries given by the crystal structures of the HIV-1RT/nevirapine complexes from protein data bank(PDB). The present calculation provides a quantummechanical interaction spectrum that explicitlyshows interaction energies between nevirapine andindividual amino-acid fragments of RT. Detailedinteractions that are responsible for drug resis-tance of two major RT mutations are elucidatedbased on computational analysis in relation to thecrystal structures of binding complexes. The presentresult provides a qualitative molecular understand-ing of HIV-1 RT drug resistance to nevirapine andgives useful guidance in designing improved inhibi-tors with better resistance to RT mutation. Proteins2005;61:423–432. © 2005 Wiley-Liss, Inc.

INTRODUCTION

HIV-1 reverse transcriptase (RT) is responsible for theconversion of single-stranded viral RNA into double-stranded DNA prior to integration into the genome of thehuman host.1 The RT is an excellent target for drug designbecause it is essential for HIV replication but is notrequired for normal cell replication. The compounds thatinhibit the DNA polymerase activity of RT can be dividedinto two broad classes: (1) the nucleoside reverse transcrip-tase inhibitors (NRTI) that inhibit viral replication byacting as chain terminators of DNA synthesis1 and (2) thenon-nucleoside reverse transcriptase inhibitors (NNRTI)that bind to a site on the RT palm sub-domain adjacent tobut distinct from the polymerase active site.2–6 Non-nucleoside inhibitors of RT are especially attractive drugcandidates because they do not function as chain termina-tors and do not bind at the dNTP site,7,8 making them lesslikely to interfere with the normal function of other DNA

polymerases and therefore less toxic than nucleoside inhibi-tors (NRTIs) such as AZT. The NNRTIs analogs such asnevirapine and Efavirenz are noncompetitive inhibitorsthat lock the polymerase active site in an inactive confor-mation and cause inhibition by allosteric modifica-tions.9–12

Although NNRTIs are highly specific and less toxic thannucleoside inhibitors, their therapeutic effectiveness islimited by relatively rapid emergence of drug-resistantHIV-1 strains. While many good inhibitors of the NNRTIclass have been reported and three, including nevirapine,are used clinically, the discovery of new, more efficaciousinhibitors is becoming increasingly important in light ofthe emergence of HIV strains that are resistant to thecurrent drugs. It is thus important to continue to designcompounds that are active against key variants thatemerge upon treatment with the inhibitors, in addition towild type. The end result of point mutations is the de-creased activity of the inhibitors, mainly due to decreasedeffect of protein–drug binding. The rapidity of the selec-tion of drug resistant HIV in patients is such that muta-tions of amino acid residues in the binding pocket of RTreduce the drug effect significantly and makes the firstgeneration NNRTIs such as nevirapine unusable in mono-therapy.13,14

An important mutation occurs for lysine to asparaginemodification for residue 103 of the RT p66 subunit (termedK103N).12,15 The activities of all three FDA approvedNNRTIs, nevirapine, delavirdine, and efavirenz (Sustiva),are diminished by the K103N mutation.16 The loss ofbinding for nevirapine by this mutant RT is 40-fold ormore.18 Another important mutation in RT is at Tyr181which gives rise to high level resistance.14,19 This Tyr181mutation has been frequently reported in resistance stud-ies for many other NNRTIs and the change is almostalways to cysteine.20 Nevirapine shows 113-fold loss ofbinding with Tyr181Cys compared to wild-type RT.17,18

Despite intensive experimental research on drug bind-ing, detailed RT-drug binding interactions and the originof mutational effects are still not fully understood. De-

*Correspondence to: John Zhang, Department of Chemistry, NewYork University, New York, NY 10003. E-mail: john.zhang@nyu.edu

Received 3 December 2004; Accepted 21 February 2005

Published online 19 August 2005 in Wiley InterScience(www.interscience.wiley.com). DOI: 10.1002/prot.20578

PROTEINS: Structure, Function, and Bioinformatics 61:423–432 (2005)

© 2005 WILEY-LISS, INC.

tailed understanding of these interactions is crucial for theanalysis and design of new RT inhibitors to make betterand mutation-resistant drug candidates. In this respect,theoretical calculation provides an attractive alternativeapproach to studies of protein–drug interaction. In particu-lar, theoretical computation of binding interaction canprovide detailed analysis of drug binding and thereforepractical guidance to the experimental design of newpotent inhibitors. Due to the large size of proteins, theoreti-cal calculation for protein interaction is limited in the levelof theory and the accuracy of the calculation. Recently,new development in quantum chemical (QC) theory hasopened possibilities for quantum chemical calculation ofprotein–drug interactions.21–23 Kuno et al.24 has recentlyreported a quantum mechanical study for RT-nevirapinebinding employing the ONIOM approaching a mixed levelof quantum treatment for the enzyme–drug system. Thisquantum study for RT-nevirapine provided importantinformation about specific bindings of nevirapine to RT.

In the present study, we perform a quantum mechanicalstudy to investigate detailed interaction mechanisms inbinding of nevirapine to HIV-1 RT wild type (WT) and twomajor single-point mutations: K103N and Y181C. The aimof our study is to help elucidate the nature of drugresistance of RT due to these mutations. The ab initiocomputation is made possible by applying a recentlydeveloped MFCC (molecular fractionation with conjugatecaps) approach.23,25,26 The MFCC approach developed forfull quantum chemical computation of protein interactionenergies is especially convenient for protein–ligand bind-ing interaction.23 In the MFCC approach, the protein orpeptide is decomposed into amino acid-based fragmentsthat are properly capped. Using this approach, computa-tion of the interaction energy between a protein andanother molecule can be carried out by separate ab initiocalculations of interaction energies between protein frag-ments and the molecule of interest. The MFCC method islinear scaling, computationally efficient, and particularlysuitable for computation on multiprocessor computer sys-tems. The MFCC method has recently been successfullyapplied to calculating interaction energies of several pro-tein–ligand systems.27–29

Due to the neglect of solvation effect, inherent limitationof the ab initio calculations at the HF and DFT levels, lackof dynamical effect, etc., the current study does not at-tempt to provide a quantitative comparision betweentheoretical calculation and the observed experimentalbinding affinities. Rather, our aim is to provide qualitativeinsight on the drug resistance of HIV-1 RT on nevirapinefrom the viewpoint of specific molecular interaction. Suchanalysis and insight could provide useful guidance indesigning new drugs with better resistance to HIV-1 RTmutations.

THEORY

In the MFCC approach to computing protein–ligandinteraction energy, the entire protein molecule is treatedby quantum mechanics in a consistent fashion.23 TheMFCC method derives its great computational efficiency

partly by foregoing some calculations of intra-proteininteraction energy and focus on intermolecular interactionenergy. In the present study, we apply the MFCC methodto compute binding interaction of nevirapine to the HIV-1RT WT and its mutations K103N and Y181C. Figure 1(A)shows the crystal structure of HIV-1 RT WT in complexwith nevirapine as given PDB (id 1VRT).30 The crystalstructures of the other two complexes are given by PDB ids1FKP (K103N)31 and 1JLB (Y181C),32 respectively. Thestructure of nevirapine is shown in Figure 1(B).

In the MFCC approach, we first decompose the wild typeHIV1 RT with 926 into 926 fragments by cutting all thebackbone peptide bonds C–N. Figure 2 shows the locationsof the cuts. At every location of cut, a pair of conjugate caps(CH3CO– and The MFCC approach developed for fullquantum chemical computation of protein interaction ener-gies is especially convenient for protein–ligand bindinginteraction.23 NHCH3) are inserted to cap the cutofffragments. In addition, there are also 925 cap speciesformed by fusing pairs of conjugate caps inserted. Usingthe MFCC approach, the expression of interaction energyfor the RT-ligand system (�EL�RT� is given by the followingexpression.23

Fig. 1. A: The crystal structure of HIV-1 RT wild type in complex withnevirapine as obtained from PDB (id 1VRT). B: Atomic geometry ofnevirapine.

424 X. HE ET AL.

EL�RT � �i�1

N

EL,Fi � �i�1

N�1

EL,CCi � �i�1

N

EFi � �i�1

N�1

ECCi � �i�1

Nd

EL,DCi

� �i�1

Nd

EDCi � �i�1

Nd

EL � EL (1)

where EL,F denotes the ligand-ith fragment energy, EL,CCi

the ligand-ith concaps (conjugate caps) energy, EFi and ECCi

are, respectively, the self energy of the ith fragment andith concaps and EL is the ligand self energy. EL,DCii is theligand–disulfide concap energy as described in Chen etal.26 The N and Nd are, respectively, the number of aminoacids and disulfide bonds. For HIV-1 RT, there are nodisulfide bonds (Nd � 0) and N � 926 for the wild typeenzyme.

In our calculations of interaction energies for RT-nevirapine complexes, we froze all heavy atoms at thecrystal structures while positions of hydrogen atoms areoptimized using the Amber program.33 Then a rigid pro-tein–ligand geometry optimization is carried to optimizethe binding structure using Amber force field (FF).34 As aresult, a slightly shifted geometry with a somewhat lowerenergy is obtained from the optimization procedure. TheMFCC ab initio calculations at both HF and DFT levelwere then carried out to generate RT-nevirapine interac-tion energies at the optimized structures. Due to computa-tional cost, however, energy minimization using MFCCcalculation was not performed.

RESULTS AND ANALYSISBinding of Nevirapine to Wild Type HIV-1 RT

The RMSD (root mean square deviation) of the opti-mized position of nevirapine within the binding complex is0.310 Å from the PDB structure (PDB id 1VRT), represent-ing only a slight deviation from its position given by thecrystal structure. Considering high computational cost inquantum chemical calculation, the MFCC calculation isperformed at the HF/3-21G level for all the residues. Inaddition, DFT B3LYP/6-31(d) calculations are performedfor selected residues with dominant binding interactionwith nevirapine. Although the absolute binding energyfrom HF/3-21G calculation is expected to be somewhatlarger, but the relative energies are quite consistent basedon our previous calculations for other protein–ligand com-plexes.28,29 Additional DFT calculations using a largerbasis set supported this conclusion. Thus, the presentMFCC calculation at HF/3-21G level shall not affect thequalitative analysis of binding interactions.

The calculated spectrum in Figure 3 shows interactionenergies between nevirapine and individual amino acidfragment (excluding caps) at geometry of the bindingcomplex. The abscissa in Figure 3 denotes the amino acidsequence of RT. The HIV-1 RT complex consists of twochains. Figure 3 shows that there is essentially no interac-tion between nevirapine and the second chain. This isfairly easy to understand from the RT-nevirapine complexstructure in Figure 1(A) where nevirapine sits quite awayfrom most part of the second chain. The interactionspectrum in Figure 3 shows that the main binding attrac-tions with nevirapine come from about six residues withbinding energies larger than 2 kcal/mol. The main bindingattractions come from four groups: the His235–Pro236group, Phe227, Tyr188, and Lys101–Lys103 group. Thebinding energies of these dominant groups with nevirap-ine are generally between 2–5 kcal/mol at HF/3-21G level.

It is worthwhile to mention that the individual fragment–nevirapine interaction energy from the MFCC calculationis “de-capped” to remove the interaction due to cap compo-nent of the fragment before the interaction spectrum isplotted. Most cap components have negligible interactionswith the inhibitor, only those cap components associatedwith strong binding fragments can have appreciable inter-action with the inhibitors. These “extra” interactions arelargely of hydrogen bonding in nature and are associated

Fig. 2. Chain representation of the protein showing locations of the cuts where concaps are introduced inthe MFCC approach.

Fig. 3. MFCC computed interaction spectrum at HF/3-21G level fornevirapine binding to HIV-1 RT wild type at complex structure. The X-axislabels amino acid number of RT and the Y-axis denotes the interactionenergy between nevirapine and the specific amino acid residue. Theeffect of caps has been properly eliminated.

DRUG RESISTANCE OF HIV-1 RT TO NEVIRAPINE 425

with the CO– and NH– groups in the concaps. Although,the MFCC scheme essentially cancels these “extra” interac-tion energies to give the correct total binding energy, it isdesirable to remove these extra energies from the indi-vidual fragment energies in order to show more clearly theinteraction spectrum. Thus, we employ an approximatescheme to remove these extra cap energies to make clearerspectra. The method is described in the Appendix.

In order to check the reliability of HF/3-21G calculation,we also calculate individual RT fragments–nevirapineinteraction energies for several dominant binding residuesusing at DFT B3LYP/6-31G(d) level. These DFT resultsare compared to those from the HF/321G calculation inTable I. As we see clearly from the table, the calculatedinteraction energies using both HF/3-21G and B3LYP/6-31G(d) method are in close agreement with each other,with the B3LYP/6-31G(d) energies being slightly higher ingeneral. Figure 4 shows the interaction spectrum com-puted using both HF/3-21G and B3LYP/6-31G(d) methodsfor eight dominant binding residues in the order of Leu100,

Lys101, Lys103(Asn103 in K103N), Tyr181(Cys181 inY181C), Tyr188, Phe227, His235 and Pro236 for RT WT,K103N and Y181C. As the figure shows, the relativespectrum is essentially unchanged from both quantumchemical calculations.

Figure 5 shows the geometric positions of six residueswhose interaction energies with nevirapine are largerthan 2.0 kcal/mol as indicated in Figure 3. The presentcalculation shows clearly that there is no strong hydrogenbond formed between nevirapine and RT. Instead, thebinding is through a number of week hydrogen bondinginteractions with binding energies about 2–5 kcal/mol.Various distances between two hydrogen atoms of nevirap-ine and oxygen atoms involved in binding interaction areshown in Figure 5. The analysis of geometry and energyindicates that these interactions are of weak hydrogenbonding in origin. The weak hydrogen binding of nevirap-ine to the Lys101–Lys103 group is important in addition tothe weak hydrogen bonding to the His235–Pro236 group,as well as a weak bonding to Phe227. The interactionspectrum in Figure 3 shows there is an attractive interac-tion between nevirapine and Tyr188 by about 2.7 kcal/mol.Geometry analysis in Figure 5 indicates that there is aweak attraction in which nevirapine donates a hydrogenatom of the aromatic ring of Tyr188 that forms a week�-hydrogen bond. Our result is consistent with previousexperimental analysis in Rizzo et al.3,35 The total interac-tion energy calculated at the HF/3-21G level is �38.10kcal/mol.

In a previous quantum study of RT-nevirapine complexusing the ONIOM method, Kuno et al.24 focused on the

TABLE I. Interaction Energies (Kcal/mol) of Nevirapine with Selected Amino Acid Fragments (Capped) of HIV-RT WTFrom MFCC Calculations at both HF/3-21G and DFT B3LYP/6-31G(d) Levels†

Fragment Lys101 Cap101 Lys102 Lys103 Tyr188 Phe227 His235 Cap235 Pro236 Cap236 Asp237

HF/3-21G �3.71 �2.64 �2.22 �2.42 �3.01 �4.01 �3.77 �5.23 �8.74 �3.69 �4.67B3LYP/6-31G(d) �2.28 �1.27 �1.34 �2.52 �2.07 �2.23 �1.89 �3.09 �5.28 �2.34 �3.21†The fragment Capi is the concaps of MeCO-NHMe inserted between the ith and i � 1th residue.

Fig. 4. The interaction spectrum computed at both HF/3-21G andB3LYP/6-31G(d) levels for eight dominant binding residues in sequenceof Leu100, Ly101, Lys103(Asn103 in K103N), Tyr181(Cys181 in Y181C),Tyr188, Phe227, His235, Pro236.

Fig. 5. The relative positions of nevirapine and HIV-1 RT WT domi-nant residues. Oxygen, nitrogen, carbon, and hydrogen atoms are in red,blue, gray, and white, respectively.

426 X. HE ET AL.

interaction of nevirapine with the aromatic ring of Tyr181.Their results using higher level MP2/6-31�G(d) calcula-tion showed that there is essentially no stacking interac-tion between their aromatic rings. Their calculation indi-cates that the suspected dispersion interaction betweenthe aromatic rings of nevirapine and Tyr181 is not impor-tant. This result is confirmed in the present calculation asthe interaction spectrum in Figure 3 shows no attractiveinteraction between nevirapine and Tyr181. Both studiesare consistent in arriving at that conclusion. The relativegeometry between nevirapine and Tyr181 is shown inFigure 6 for reference.

Our calculation does not show any strong hydrogen bondformed between nevirapine and the wild type HIVRT. In-stead, a number of weak hydrogen bonding interaction existsbetween them. Of course, this could also be understood fromthe geometries shown in Figure 5 where no single optimalhydrogen bond distance exists. The fact that no single stronghydrogen bonding exists between nevirapine and HIV-1 RTrenders the drug susceptible to drug-resistance by mutationas will be shown in the following studies.

Binding of Nevirapine to K103N Mutant

We use the crystal structure from PDB (id 1FKP) to studynevirapine binding to K103N mutation of HIV-1 RT. Follow-ing the same procedures described in the previous section fornevirapine binding to wild type HIV-1 RT, the RMSD of theoptimized complex structure is 0.278 Å from the experimen-tal geometry. The MFCC calculation is then performed togenerate the interaction spectrum. The spectrum is com-puted at the HF/3-21G level while a selected number ofindividual interacting fragments are computed at DFTB3LYP/6-31G(d) level for verification of the results.

Figure 7 shows the K103N-nevirapine interaction spec-trum. Here we find only three major attractive interac-

tions between nevirapine and K103N with binding ener-gies greater than 2 kcal/mol as shown in Figure 7. HereLys101 becomes the dominant binding partner to nevirap-ine followed by Pro236 and Phe227. Comparing with theinteraction spectrum for the wild type in Figure 3, wenotice that binding of the nevirapine to the His235-Pro236group is significantly weakened in K103N than in the wildtype. In addition, the �-hydrogen bonding of nevirapine toTyr188 is also greatly diminished.

From the interaction spectrum in Figure 7 we find thatthe attractive interaction of nevirapine with Lys101 groupis actually stronger than that with the wild type (cf. Fig. 3).The result can be easily explained from the complexstructure in Figure 8. Here we see that the distancebetween the hydrogen atom of nevirapine and oxygen atomof the Lys101 group is reduced to 2.32 Å as compared to2.56 Å in the wild type. This reduction in distance resultedin a somewhat tighter binding of nevirapine to Lys101compared to that in the wild type. However, Lys103 is now

Fig. 6. The relative positions of nevirapine and Tyr181 in complex ofHIV-1 RT wild type in which some interatomic distances are shown.

Fig. 7. Similar to Figure 3 except for the interaction spectrum ofnevirapine with K103N mutant.

Fig. 8. Similar to Figure 5 except for the K103N mutant.

DRUG RESISTANCE OF HIV-1 RT TO NEVIRAPINE 427

replaced by Asn103 in K103N as shown in Figure 8. Thismutation resulted in the loss of binding to the originalLys103 as can be seen from the spectrum in Figure 7.

The strengthening of nevirapine binding to Lys101 is,however, at the expense of having weaker attractiveinteractions with the main binding group of His235-Pro236. Here we see that the distances between hydrogenatoms of nevirapine and oxygen atoms of the His235-Pro236 group have increased by an average of about 0.5 Å,resulting in a weak hydrogen bonding attraction betweennevirapine and Pro236. In fact the hydrogen distancebetween nevirapine and His235 is increased from 2.77 to3.72 Å, resulting in the loss of binding to His235 in K103N.

The loss of the weak �-hydrogen bonding betweennevirapine and the aromatic ring of Tyr188 is a result ofconformational change of the complex. Although the dis-tance of the donor hydrogen atom of nevirapine to thearomatic ring of Tyr188 remains essentially constant, thehydrogen atom is now positioned further off-center towardthe aromatic ring of Tyr188 relative to that in the wild typeas shown in Figure 9. This results in the essential loss ofthis weak �-hydrogen bonding. Thus the structural changeof the K103N-nevirapine complex resulted in overall weakbinding by nevirapine. The overall interaction energy toK103N is �23.88 kcal/mol which is about 14.2 kcal/molless than that to the wild type. This is qualitativelyconsistent with the observed 40-fold decrease of nevirap-ine binding to K103N mutant.

The energies of these dominant binding interactionsfrom both HF and DFT calculations are listed in Table IIfor comparison.

Binding of Nevirapine to Y181C Mutant

Following the same procedure as before, the optimizedcomplex structure of Y181C-nevirapine is obtained withan RMSD of 0.198 Å from the experimental structure(PDB id 1JLB). The MFCC calculation for Y181C-nevirap-ine complex is performed and the interaction spectrum isgenerated at HF/3-21G level.

The interaction spectrum in Figure 10 shows threeresidues that have attractive interaction energy of over 2kcal/mol with nevirapine. The three dominant attractiveinteractions come from Lys101, Lys103, and Phe227.Similar to the case of K103N, nevirapine binding toLys101 becomes dominant. The residue Lys103 has over2-kcal/mol attractive interaction with the drug while bind-ing to Pro236 is significantly weakened in Y181C mutant.

The conformational change of the binding complex ofY181C-nevirapine is somewhat similar to that of K103N–nevirapine. The major binding group His235–Pro236 movedaway from nevirapine and thus results in a significantweaker attraction between nevirapine and this group.Compared to K103N, nevirapine moved further away fromthis binding group. Figure 11 shows that the hydrogen–oxygen distance between nevirapine and Pro236 is furtherincreased from 3.11 to 3.9 Å, resulting in a significant lossof attraction between nevirapine and Pro236. On the otherhand, the hydrogen–oxygen distance between nevirapineand Lys101 is 2.47 Å, which is shorter than 2.56 Å in wild

type but longer than 2.32 Å in K103N. This explains thatthe calculated binding attraction between nevirapine andLys101 is in between that in wild type and K103N.

This conformational change explains the reason whynevirapine have tighter binding with Lys101 in bothmutants than in the wild type. Also, the loss of binding toTyr188 is due to the same conformational change thatresults in an off-center positioning of the donor hydrogenatom toward the aromatic ring of Tyr188 as shown inFigure 9.

An important result from the present calculation is thatthere are strong repulsive interactions between nevirap-ine and the Y181C mutant. First, the conformationalchange in Y181C complex brought Leu100 close to nevirap-ine as shown in Figure 11. This results in a repulsion ofabout 2.5 kcal/mol. In both wild type and K103N, Leu100is away from nevirapine. In addition to repulsive interac-tion with Leu100, there is also a strong repulsion due tocollision of nevirapine with the side chain of the mutated

Fig. 9. The relative position of Tyr188 residue in HIV-1 RT complexedwith nevirapine for (A) wild type, (B) K103N mutant, (C) Y181C mutant.

428 X. HE ET AL.

Cys181. The 7.8-kcal/mol repulsive energy shown in Fig-ure 10 is rather significant. Figure 12 plots the relativegeometric positions of nevirapine with Cys181. As seenfrom the figure, the distance between the sulfur atom inthe side chain of Cys181 and the nearest carbon atom ofnevirapine is only 3.16 Å. This is the main cause of strongvan der Waals repulsion between nevirapine and themutated Cys181 residue. As a direct result of this strong

repulsion, the binding energy of nevirapine to Y181Cmutant is reduced to �15.70 kcal/mol compared to �38.10kcal/mol for the wild type and �23.88 for K103N mutant.The result is also qualitatively consistent with the experi-mental finding that Y181C mutant causes a 113-foldreduction in nevirapine binding, compared to a 40-foldreduction in binding to K103N mutant. Table III lists thedominant interaction energies between nevirapine andY181C fragments computed at HF/3-21G and B3LYP/6-31G(d) levels for comparison.

In all three RT–nevirapine complexes, the attractiveinteraction between nevirapine and Phe227 is almostconserved, only slightly weakened in mutations. For easycomparison, we list in Table IV the interaction energiesbetween nevirapine and dominant amino-acid fragmentsof all three RTs computed at HF/3-21G and DFT B3LYP/6-31G(d) level. The computed total binding energies atHF/3-21G level are given in Table V, together with thosefrom Amber force field calculation. Although the absoluteinteraction energy could not be directly related to bindingaffinity, the relative interaction energy among differentmutants should provide useful information about drugresistance or mutational effect. The gas-phase interactionenergies listed in Table V show qualitative correlationwith the experimentally observed binding loss of nevirap-ine in two mutants. The effect of mutation from quantumcalculation is larger and more sensitive than AMBER forcefield result as shown in Table V.

DISCUSSION AND CONCLUSION

In this study, the MFCC method is applied to calculatinginteraction energies of FDA approved nevirapine bindingto HIV-1 RT and two of its major mutants, K103N andY181C. The enzyme– nevirapine complex contains over

TABLE II. Similar to Table I But for HIV-RT K103N Mutant

Fragment Lys101 101Cap Lys102 Phe227 235Cap Pro236 Asp237

HF/3-21G �5.88 �4.57 �3.44 �2.59 �2.64 �4.46 �2.44B3LYP/6-31G(d) �3.42 �2.15 �1.46 �1.80 �2.04 �3.26 �2.13

Fig. 10. Similar to Figure 3 except for the interaction spectrum ofnevirapine with Y181C mutant.

Fig. 11. Similar to Figure 5 except for the Y181C mutant.

Fig. 12. The relative position of Cys181 residue in Y181C mutantcomplexed with nevirapine.

DRUG RESISTANCE OF HIV-1 RT TO NEVIRAPINE 429

15,000 atoms and quantum MFCC computation is carriedout at HF/3-21G and DFT B3LYP/6-31G(d) levels. Thecomputational results using both methods are consistentwith each other. By employing the MFCC method, we canstudy protein–inhibitor binding mechanism using quan-tum mechanical calculations. Our calculated interactionenergies for RT/nevirapine complexes provide a qualita-tive explanation on experimentally observed large reduc-tion in nevirapine binding to the enzyme upon mutationsto K103N and Y181C.

The followings are specific findings.

1. There is no strong hydrogen bonding of nevirapine toHIV-1 RT. The binding mechanism is due to collectiveeffect of several weak hydrogen bonding interactions.The His235–Pro236 is the dominant group in nevirap-ine binding to the wild type followed by the Lys101–103group, Phe227 and a �-hydrogen bonding to Tyr188. Noattractive stacking interaction between nevirapine andTyr181 exists.

2. Protein conformational change due to mutation causesnevirapine to move away from its major binding groupHis235–Pro236 in RT and results in the weakening ofhydrogen bonding interaction. This effect is present inboth K103N and Y181C mutations. In both mutants,

Lys101 becomes the dominant attractive residue, andweak �-hydrogen binding to Tyr188 is diminished dueto conformational change that results in an off-centerposition of the donor hydrogen atom. However, bindingto Phe227 is more or less conserved in both mutations.

3. Binding of nevirapine to Y181C mutant is furtherhindered by a van der Waals repulsion between nevirap-ine and the sulfur atom in the side chain of the mutatedCys181 residue, as well as a relatively weak repulsionbetween nevirapine and Leu100.

A fundamental reason for the significant loss of bindingability of the first generation drug nevirapine to mutantsof HIV-1 RT is the fact that there are no strong hydrogenbonds between the drug and the enzyme. Thus, conforma-tional change of the enzyme–drug complex due to muta-tion can easily weaken the weak binding interactions.Thus, it is desirable to introduce strong hydrogen bondingto the conservative residues of the enzyme by modifyingthe inhibitor. The quantum interaction energy spectrumbased on the MFCC method is a powerful tool for detailedanalysis of drug binding mechanism at quantum mechani-cal level. Thus it should play an important role in structure-based drug design.

While ab initio calculation at HF level ignores the correla-tion energy, DFT method like B3LYP does include electroncorrelation energy. Although both methods employed onlyground state electron wavefunction and therefore do not givecorrect dispersion energy, they both give reasonably reliableenergy profiles and molecular structures and are widely usedin practical ab initio calculations of polyatomic systems. Thenext level of ab initio calculation is at MP2 level but it will bemore computationally expensive, however.

Some discussion is in order to address the issue ofsolvation and protein conformational change. It is impor-tant to note that the present study focuses on the analysisof specific binding interaction between the ligand and the

TABLE III. Similar to Table I But for HIV-RT Y181C Mutant

Fragment Leu100 Lys101 101Cap Lys102 Lys103 Cys181 Tyr188 Phe227 235Cap Pro236

HF/3-21G 2.02 �5.05 �3.72 �2.63 �2.31 9.21 �2.37 �2.76 �2.25 �3.05B3LYP/6-31G(d) 2.81 �3.08 �1.83 �1.25 �2.38 7.63 �1.35 �1.46 �1.78 �2.41

TABLE IV. Interaction Energy (kcal/mol) Between Nevirapine and Amino Acid Fragments as well as Comcaps Caps forthe Wild Type, K103N and Y181C Mutants of HIV-1 RT at both HF/3-21G and DFT B3LYP/6-31G(d) Levels

Fragment

Wild type K103N Y181C

HF/3-21G B3LYP/6-31G(d) HF/3-21G B3LYP/6-31G(d) HF/3-21G B3LYP/6-31G(d)

Leu100 �1.19 �0.58 �0.57 0.31 2.02 2.81Lys101 �3.71 �2.28 �5.88 �3.42 �5.05 �3.08Lys102 �2.22 �1.34 �3.44 �1.46 �2.63 �1.25Lys103 �2.42 �2.52 0.92(Asn) 0.53(Asn) �2.31 �2.38Tyr181 0.75 1.34 �0.32 0.07 9.21(Cys) 7.63(Cys)Tyr188 �3.01 �2.07 0.45 0.70 �2.37 �1.35Phe227 �4.01 �2.23 �2.59 �1.80 �2.76 �1.46His235 �3.77 �1.89 �1.24 �0.84 �0.70 �0.64Pro236 �8.74 �5.28 �4.46 �3.26 �3.05 �2.41Asp237 �4.67 �3.21 �2.44 �2.13 �0.43 �0.69

TABLE V. Calculated Interaction Energies of NevirapineBinding to Wilde Type and Mutants of HIV-1 RT†

WT K103N Y181C

MFCC (HF/3-21G)(kcal/mol)

�38.1 �23.9 �15.7

Amber (parm99)(kcal/mol)

�55.9 �44.1 �43.6

For reference, the experimental binding loss of nevirapine to HIV-1 RTis, respectively, 40-fold and 113-fold to K103N and Y181C muta-tions.17,18

430 X. HE ET AL.

protein components only and the calculation is based onthe experimental crystal structures. No attempt is made tocalculate binding affinity or free energy of binding. Sincecrystal structures represent the most reliable structures ofthe binding complex (although in the average sense), thecomputed protein–ligand interaction should be meaning-ful and not seriously affected by considering solvation andsmall conformational change using, e.g., molecular dynam-ics simulation. Another issue concerns the charged resi-dues on the surface protein. Since the ligand is neutral, ourcalculated interaction spectrum shows clearly that thedominant binding interaction is between the ligand and afew amino acids of the protein that are located in or nearthe binding pocket, which is quite hydrophobic. No signifi-cant long range interaction between the ligand and thecharged protein residues on the surface of the protein isfound. Thus, the general conclusion should not be seri-ously impacted by the inclusion of solvent on the surface ofthe protein. The result of our QM calculation does showqualitative agreement with the FF calculation with somequantitative difference for certain interactions. However,because the level of current ab initio calculation is rela-tively low and no dispersion energy is obtained, quantita-tive comparison between the FF and QM interactionenergy is not very meaningful. However, work is inprogress to use higher level ab initio methods to do thesecalculation in order to perform meaningful quantitativecomparison between QM and FF energies.

The current study provides theoretical insight from theviewpoint of specific molecular interaction on the drug resis-tance of HIV-1 RT to nevirapine. Work is in good progress tointroduce modification of nevirapine to enhance its resis-tance to RT mutations based on the present analysis.

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APPENDIX

Here we describe an approximate method to remove theextra interaction energy between the ligand and the capcomponent of the protein fragment from the MFCC calcula-

tion. First we use Amber force field to calculate the interac-tion energy between the ligand and each half of the ithconcaps (CH3CO� and �NHCH3 defined as EL�CH3COi

c andEL�NHCH3i

c , respectively. We then use these energies to calcu-late the ratios �i and i for both of the ith conjugate caps,CH3CO– and –NHCH3. These ratios are defined as

�i �EL�CH3COi

c

EL�CH3COic � EL�NHCH3i

c (2)

i � 1 � �i (3)

We use these ratios to partition the computed MFCCinteraction energy between nevirapine and each pair of ithconcaps into their corresponding residue fragment tocancel the extra energy due to cap component. The ithcapped protein fragment is in the form of (CH3CO–Ri–NHCH3). In the MFCC Equation 1, EL,Fi denotes theenergy of the ligand and the ith fragment, EL,CCi denotesthe energy of the ligand and the ith concaps. Now we useERi to denote the interaction energy between the ligandand the uncapped ith fragment.

ERi � EL,Fi � �i�1EL,CCi�1 � iEL,CCi (4)

Using ERi instead of EL,Fi the interaction spectrum showsreliably direct interaction energies between nevirapine andthe residues of the enzyme without the extra cap effect.

432 X. HE ET AL.