Structural mechanisms of drug resistance for mutations at codons 181 and 188 in HIV-1 reverse transcriptase and the improved resilience of second generation non-nucleoside inhibitors1

doi:10.1006/jmbi.2001.4988 available online at http://www.idealibrary.com on

J. Mol. Biol. (2001) 312, 795±805

Structural Mechanisms of Drug Resistance for Mutations at Codons 181 and 188 in HIV-1 Reverse Transcriptase and the Improved Resilience of Second Generation Non-nucleoside Inhibitors J. Ren1, C. Nichols1, L. Bird1, P. Chamberlain1, K. Weaver2, S. Short2 D. I. Stuart1,3 and D. K. Stammers1,3* 1

Structural Biology Division The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive Oxford OX3 7BN, UK 2 Glaxo Smith Kline, 5 Moore Drive, Research Triangle Park NC 27709, USA 3

Oxford Centre for Molecular Sciences, New Chemistry Building, South Parks Road Oxford OX1 3QT, UK

Mutations at either Tyr181 or Tyr188 within HIV-1 reverse transcriptase (RT) give high level resistance to many ®rst generation non-nucleoside inhibitors (NNRTIs) such as the anti-AIDS drug nevirapine. By comparison second generation inhibitors, for instance the drug efavirenz, show much greater resilience to these mutations. In order to understand the structural basis for these differences we have determined a series of seven crystal structures of mutant RTs in complexes with ®rst and second generation NNRTIs as well as one example of an unliganded mutant RT. Ê , Tyr181Cys RT (efavirenz) to These are Tyr181Cys RT (TNK-651) to 2.4 A Ê , Tyr181Cys RT (nevirapine) to 3.0 A Ê , Tyr181Cys RT (PETT-2) to 2.6 A Ê , Tyr188Cys RT (nevirapine) to 2.6 A Ê , Tyr188Cys RT (UC-781) to 3.0 A Ê and Tyr188Cys RT (unliganded) to 2.8 A Ê resolution. In the two pre2.6 A viously published structures of HIV-1 reverse transcriptase with mutations at 181 or 188 no side-chain electron density was observed within the p66 subunit (which contains the inhibitor binding pocket) for the mutated residues. In contrast the mutated side-chains can be seen in the NNRTI pocket for all seven structures reported here, eliminating the possibility that disordering contributes to the mechanism of resistance. In the case of the second generation compounds efavirenz with Tyr181Cys RT and UC-781 with Tyr188Cys RT there are only small rearrangements of either inhibitor within the binding site compared to wild-type RT and also for the ®rst generation compounds TNK-651, PETT-2 and nevirapine with Tyr181Cys RT. For nevirapine with the Tyr188Cys RT there is however a more substantial movement of the drug molecule. We conclude that protein conformational changes and rearrangements of drug molecules within the mutated sites are not general features of these particular inhibitor/mutant combinations. The main contribution to drug resistance for Tyr181Cys and Tyr188Cys RT mutations is the loss of aromatic ring stacking interactions for ®rst generation compounds, providing a simple explanation for the resilience of second generation NNRTIs, as such interactions make much less signi®cant contribution to their binding. # 2001 Academic Press

*Corresponding author

Keywords: HIV-1; reverse transcriptase mutants; drug resistance mechanism; X-ray crystallography; DNA polymerase

Introduction Abbreviations used: HIV-1, human immunode®ciency virus type-1; RT, reverse transcriptase; NNRTI, nonnucleoside RT inhibitor; NRTI, nucleoside analogue RT inhibitor. E-mail address of the corresponding author: [email protected] 0022-2836/01/040795±11 $35.00/0

HIV-1 reverse transcriptase (RT) is a p66/p51 heterodimer that copies the genomic RNA of the virus into proviral DNA which subsequently becomes integrated into the host cell DNA. This key role in the replicative cycle of the virus has made RT the target for the development of many # 2001 Academic Press

796

HIV-1 RT Drug Resistance Mutations

anti-HIV drugs. These drugs fall into two classes: (i) the nucleoside analogue inhibitors (NRTIs) which, in their triphosphate form, bind at the polymerase active site and after incorporation into the primer strand cause termination of the DNA chain; (ii) the non-nucleoside inhibitors (NNRTIs), which are generally speci®c for HIV-1 RT and bind at an Ê from the active allosteric site approximately 10 A site causing a displacement of the catalytic aspartate residues.1,2 The introduction of multi-drug regimens consisting of various combinations of NRTIs, NNRTIs as well as protease inhibitors (PIs) to treat HIV infection has been a major therapeutic advance in the ®ght against AIDS.3 However, the selection of HIV with resistance to various drugs appears to be an inevitable consequence of the rapid turnover of HIV together with the chronic

So-called second generation NNRTIs such as the anti-HIV drug efavirenz as well as the experimental compound UC-781 (Scheme 1) retain much higher potencies in the presence of a range of single-point mutations.12,13 For example whilst nevirapine shows 113-fold loss of binding with Tyr181Cys compared to wild-type RT, efavirenz shows only a 2.5-fold reduction in binding to this mutant RT.12,14 With the mutation at codon 188, nevirapine shows an 83-fold reduction in af®nity for Tyr188Cys RT compared to wild-type RT whilst for UC-781 there is a ®vefold reduction in potency for the Try188His mutation.13,14 Both Tyr181 and Tyr188 are positioned at the ``top'' of the NNRTI binding site, forming in conjunction with the highly conserved residue Trp229, a hydrophobic sub-pocket.15 ± 17

Scheme 1

nature of the infection which requires long-term drug therapy.4,5 The rapidity of the selection of drug resistant HIV in patients was such that single-point mutations in the virus made ®rst generation NNRTIs such as nevirapine unusable in monotherapy.6,7 Amongst the mutations in RT that were originally described for nevirapine resistance were those at Tyr181 and Tyr188, both of which gave rise to high level resistance.7,8 Mutation at Tyr181 has since been frequently reported in resistance studies for many other NNRTIs and the change is almost always to cysteine.5 In the case of the codon 188 mutation, a greater variety of changes are reported, nevirapine and HEPT select the Tyr188Cys mutation,8,9 whereas Tyr188His or Tyr188Leu are selected with TIBO or a-APA.10,11

Crystal structures of wild-type RT with NNRTIs used in this current work such as nevirapine, TNK651, PETT-2 (Scheme 1) or with other ®rst generation compounds show extensive stacking interactions of the side-chains of Tyr181 and Tyr188 with the aromatic rings of the inhibitors.15-21 Structures have been reported for RT (Tyr181Cys) Ê with the ®rst generation inhibitor Cl-TIBO at 3.2 A 22 resolution and RT (Tyr188Leu) with the second Ê generation compound HBY097 at 3.3 A resolution;23 in both cases no electron density was observed for the mutated side-chains within the NNRTI binding site, this being attributed to disordering. It was not clear whether such putative disordering of the mutant side-chains was a general feature of RTs with mutations at codons 181 or 188 and to what extent this could be a general contri-

797

HIV-1 RT Drug Resistance Mutations

bution to the mechanism of resistance for such mutants. To address systematically the interplay between the two resistance mutants and different generations of NNRTIs we have determined the structures of both mutant enzymes with a range of inhibitors. We have been able to achieve suf®cient resolution to allow full re®nement of most complexes, thereby clarifying the structural mechanisms of resistance.

Results Overall RT structure We have determined and re®ned the structures of a series of seven distinct HIV-1 RT mutant (either Tyr181Cys or Tyr188Cys)/inhibitor comÊ, plexes: Tyr181Cys RT (TNK-651) to 2.4 A Ê , Tyr181Cys RT Tyr181Cys RT (efavirenz) to 2.6 A Ê , Tyr181Cys RT (PETT-2) to (nevirapine) to 3.0 A Ê , Tyr188Cys RT (nevirapine) to 2.6 A Ê, 3.0 A Ê and Tyr188Cys Tyr188Cys RT (UC-781) to 2.6 A Ê . Details of the structure RT (unliganded) to 2.8 A determinations and the re®nement statistics are shown in Table 1. Omit maps clearly show electron density for both the mutated side-chain in the p66 subunit for each of the structures as well as for the bound NNRTI where appropriate (Figures 1 and 2). Electron density is also clearly visible for the mutated residues within the p51 subunit (data not shown). In the case of the Tyr188Cys RT (nevira-

pine) complex the inhibitor is probably not at full occupancy which is re¯ected in the rather high B-factors for the nevirapine. Previous reports have described the structures of the corresponding inhibitor complexes of wild-type HIV-1 RT, with Ê ,19 PETT-2 Ê ,16 TNK-651 at 2.5 A nevirapine at 2.2 A 20 24 Ê Ê at 3.0 A, UC-781 at 2.9 A and efavirenz at Ê .25 These wild-type structures are used below 2.5 A in comparisons with mutant RTs. As expected the overall fold and gross structural features of the HIV-1 RT heterodimer are unchanged in all structures. However we do observe movement of ¯exible regions of the RT molecule, particularly the b-9 to b-11 sheet, as well as repositioning of inhibitor molecules and side-chains in the mutated drug pockets for some of the structures as described below. Comparison of wild-type and Tyr181Cys RT mutant structures Nevirapine is located in almost exactly the same position in both the wild-type and mutant enzymes (Figure 3(a)). There are however some slight perturbations of the protein. The reduced bulk of the mutant side-chain apparently allows Tyr188 to move closer towards it. This in turn causes Trp229 to re-orientate, which has the knockon effect of causing a rearrangement of the b-9 to b-11 sheet region with side-chain movements extending to Tyr318. In the case of TNK-651 there

Figure 1. Simulated annealing omit electron density maps showing the Cys181 and bound inhibitors at the NNRTI pocket of Tyr181Cys mutant HIV-1 RT. (a) Nevirapine, (b) TNK-651, (c) PETT-2 and (d) efavirenz.

3.11-3.0 2175 94.9 0.9

30-3.0 21,733/1111 0.205/0.250 0.195 7821/20/0.012 1.7 81/87/78/3.8

B. Outer resolution shell Ê) Resolution range (A Unique reflections Completeness (%) Average I/s(I)

C. Refinement statistics Ê) Resolution range (A No. of reflections (working/test) R-factorc (Rwork/Rfree) R-factorc (all data) No. of atoms (protein/inhibitor/water) Ê) rms bond length deviation (A rms bond angle deviation (deg.) Ê 2)d Mean B-factor (A Ê 2) rms backbone B-factor deviation (A 30-2.5 37,727/1989 0.204/0.275 0.200 7819/27/46 0.008 1.4 55/62/43/47 4.0

2.59-2.5 3890 98.1 0.9

Y181C-TNK-651 KEK BL-6A Fuji BAS III 1.000 140.4, 111.2, 73.0 30-2.5 184,430 40,014 99.2 7.3 0.156 40

b

Rmerge ˆ jI ÿ hIij/hIi. Determined from TRUNCATE in the CCP4 suite.44 c R-factor ˆ jFo ÿ Fcj/Fo. d Mean B-factor for main-chain, side-chain, inhibitor and water atoms, respectively.

a

Y181C-nevirapine KEK BL-6A Fuji BAS III 1.000 141.2, 110.3, 73.3 30-3.0 91,509 22,958 97.3 7.5 0.132 67

A. Data collection details Data set Data collection site Image plate Ê) Wavelength (A Ê) Unit cell dimensions (a, b, c in A Ê) Resolution range (A Observations Unique reflections Completeness (%) Average I/s(I) Rmergea Ê 2)b B-factor from Wilson plot (A

Table 1. Statistics for crystallographic structure determinations

30-3.0 21,319/1088 0.225/0.281 0.213 7689/23/0.011 1.6 84/90/73/3.8

3.11-3.0 2205 97.0 0.8

Y181C-PETT-2 KEK BL-6A Fuji BAS III 1.000 139.2, 109.3, 73.5 30-3.0 80,792 22,461 97.0 8.7 0.112 82

30-2.5 36,661/1930 0.236/0.302 0.229 7783/21/123 0.007 1.5 71/76/54/52 3.7

2.59-2.5 3769 97.4 1.2

Y181C-efavirenz ESRF ID14-3 MAR CCD 0.9310 138.5, 109.0, 73.2 30-2.5 207,449 38,709 98.7 20.5 0.069 72

30-2.8 24,712/1313 0.262/0.337 0.249 7656/-/24 0.012 1.7 88/94/-/39 4.0

2.9-2.8 2234 81.9 1.6

Y188C-APO ESRF ID2 MAR 345mm 0.9903 137.8, 109.5, 72.9 30-2.8 97,640 26,234 94.1 14.1 0.073 67

20-2.6 32,046/1674 0.237/0.303 0.226 7750/20/60 0.009 1.5 78/84/98/59 5.8

2.69-2.6 3335 99.6 1.1

Y188C-nevirapine ESRF ID14-2 MAR CCD 0.933 137.3, 109.2, 72.0 20-2.6 220,241 33,769 99.3 17.3 0.070 71

30-2.6 32,017/1673 0.220/0.291 0.214 7680/22/43 0.009 1.5 69/74/47/44 6.3

2.69-2.6 3210 92.9 1.2

Y188C-UC-781 ESRF BM14 MAR 345mm 0.918 138.0, 109.8, 73.0 30-2.6 94,725 33,717 95.9 9.6 0.061 68

799

HIV-1 RT Drug Resistance Mutations

Figure 2. Simulated annealing omit electron density maps showing the Cys188 and bound inhibitors at the NNRTI pocket of Tyr188Cys mutant HIV-1 RT. (a) Unliganded, (b) nevirapine, and (c) UC-781. Cys188 is oxidised in (a) and (c).

is a rearrangement of the inhibitor's propyl group in response to the loss of bulk of the adjacent Tyr181Cys mutation, the pyrimidine ring of the inhibitor does not shift relative to its position in the wild-type complex and the hydrogen bond to the main-chain carbonyl group of Lys101 is maintained (Figure 3(b)). The side-chain of Tyr188 maintains its position and hence the conformation of Trp229 is the same as for the wild-type enzyme, such that there are only small conformational rearrangements of the b-9 to b-11 sheet. For PETT2 as with TNK-651 rearrangement of the inhibitor conformation and position is small and occurs at parts of the inhibitor that are proximal to the Tyr181Cys mutation (Figure 3(c)). The side-chain of Tyr188 swings towards Cys181 but this is not suf®cient to cause the ¯ip of the Trp229 side-chain as is seen for nevirapine presumably because the side-chain is positioned slightly higher in the pocket than for the nevirapine complex. Perturbations of the b-9 to b-11 sheet are thus small although some changes in Phe227 and Leu234 are seen in the mutant structure. Efavirenz is a relatively rigid molecule, unable to undergo signi®cant conformational rearrangement, which also moves little in the Tyr181Cys complex compared to wild-type (Figure 3(d)). The cyclopropyl group is less bulky than the corresponding aromatic rings for the other inhibitors investigated here, which allows Trp229 to ¯ip. Comparison of wild-type and Tyr188Cys RT mutant structures When bound to Tyr188Cys RT, nevirapine Ê towards the undergoes a shift of up to 0.9 A mutated residue (Figure 4(b)). The movement is towards the b-9 to b-11 sheet, which is in turn displaced as a result. A consequence of the movement of nevirapine is that the side-chains of Glu138 (from the p51 subunit) and Leu100 located on the other side of the NNRTI pocket from residue 188

track the movement of the inhibitor. The Tyr188Cys mutation produces a smaller overall shift in the position of UC-781 than nevirpaine although the furan ring is displaced by a 30  rotation (Figure 4(c)). The unliganded structure of Tyr188Cys reveals the expected collapse of the NNRTI pocket, in a manner very similar to that observed for the wild-type enzyme. The conformation of b-9 to b-11 strands appear to be even more ¯exible in the absence of bound NNRTI and differ slightly in conformation (Figure 4(a)). For the UC-781 RT structure as well as the unliganded Tyr188Cys RT mutant structure, there is additional electron density associated with the sulphur atom of the cysteine side-chain. We have previously noted similar additional density in the case of Cys280, which was modelled as two oxygen atoms, presumed to be the result of sulphydryl oxidation.26 The Cys188 was similarly modelled for the unliganded and UC-781 complex.

Discussion The analyses including examples of ®rst and second generation NNRTIs in complexes with either Tyr181Cys or Tyr188Cys HIV-1 RTs provide insights into the structural mechanisms of resistance for these clinically signi®cant HIV drug resistance mutations. We have been able to determine structures at higher resolution than previously obtained for these RTs mutated at codons 181 and 188, which has allowed full re®nement in most cases, thereby giving some con®dence in the results obtained. Co-crystallisation of even weak binding ®rst generation compounds such as nevirapine (af®nity 100-fold reduced for Tyr181Cys and Tyr188Cys mutants) with mutant RTs demonstrates that nevirapine has suf®ciently slow off-rates from the mutant RT complexes to allow crystallisation of these enzyme-inhibitor complexes.27

800

HIV-1 RT Drug Resistance Mutations

Figure 3 (legend shown opposite)

HIV-1 RT Drug Resistance Mutations

In all four Tyr181Cys mutant RT structures reported here, the cysteine side-chains are clearly visible in omit electron density maps in each case. This is in contrast to the previously described structure for the Tyr181Cys RT complex with ClTIBO where no density was observed for the mutated residue within the NNRTI binding site and it was concluded that the side-chain was disordered.22 Similarly no density was observed for the Tyr188Leu mutant side-chain within the NNRTI binding site in the structure reported by Hsiou et al.23 Whilst reduction in the interactions of NNRTIs could be observed with RT mutated at Tyr181 and Tyr188 in these earlier studies, whether the disordering of the mutated side-chains was a general feature of such mutations was not clear.22,23 It may be that the apparent discrepancy of the earlier analyses22,23 with the results reported here could re¯ect the lower resolution of those earlier analyses, which precluded a full structural re®nement. In the case of the complex of Tyr188Cys RT with UC-781 and the unliganded Tyr188Cys RT there was evidence of additional density associated with the cysteine sulphur atom which we ascribe to oxidation as has previously been observed for Cys280.16 For the Tyr188Cys RT complex with nevirapine the cysteine side-chain is not oxidised however. The question arises as to what extent the presence of a modi®ed cysteine residue alters any interpretation of the results reported here. We note that a variety of resistance mutations can be accommodated at the 188 position including leucine and histidine. In terms of bulk the modi®ed cysteine residue we observe is close to leucine. Our new set of structural data allows us to dissect out factors that could account for the difference in resilience between ®rst and second generation NNRTIs when confronted by mutations at codons 181 or 188. We have previously described three factors that could contribute to the greater resilience of second generation inhibitors such as efavirenz.25 These are (i) the nature of the interactions e.g. main-chain hydrogen bonding which is less subject to the effects of side-chain mutation, (ii) overall size of the compound and (iii) the ability to rearrange and adapt to a mutated NNRTI pocket. In the case of efavirenz and the Lys103Asn mutation in RT we showed that the inhibitor was repositioned within a somewhat restructured NNRTI pocket, giving rise to signi®-

801 cant differences in side-chain contacts for the drug with mutant RT. Arnold and colleagues postulated that ¯exibility was an important feature for the second generation drug HBY097 in maintaining its interaction with Tyr188Leu RT.23 They reported changes in the conformation of the HBY097 inhibitor itself as well as rearrangement of the side-chain of Phe227 within the NNRTI pocket of the mutant Tyr188Leu RT, resulting in some different protein contacts with the inhibitor. Our present results reveal a fairly straightforward story for the effect of the Tyr181Cys and Tyr188Cys upon the binding of ®rst and second generation NNRTIs to RT. In summary ®rst generation compounds are more dependent on ring stacking interactions so lose more binding energy with the mutant which they are unable to reclaim by rearrangement. In the case of the second generation compound UC-781 bound to Tyr188Cys RT, apart from a rotation of the methyl-furan ring, the inhibitor-protein contacts are largely the same as for the wild-type RT, whereas nevirapine shifts within the binding site, giving a greater change in contacts. However this movement is clearly unable to compensate for the loss of ring stacking interactions with the wild-type Tyr188 (UC-781 only contacts Tyr188 via its aliphatic dimethylallyl group). Aromatic ring stacking with Tyr181 is also a signi®cant contribution to the binding energy of nevirapine to wild-type RT. There is minimal rearrangement of nevirapine in the Tyr181Cys RT and hence loss of ring stacking accounts for most of the 100-fold reduction in binding. Efavirenz, a second generation NNRTI, interacts with the Tyr181 only via its propenyl cycopropyl group Ê rather than via with a closest approach of 3.7 A the extensive contacts of a pyridine ring in the case of nevirapine. The minimal loss of interaction for the Tyr181Cys mutant RT results in little displacement of the efavirenz. Flexibility of the relatively rigid efavirenz molecule itself is unlikely to be a signi®cant factor in explaining resilience (as has been proposed in the case of HBY09723). In other examples of second generation compounds such as S-1153, an aromatic ring is located in the Tyr181, Tyr188, Trp229 subpocket. S-1153 has a 2,5 dichloro-phenyl group, which we have shown to form more extensive contacts with the Trp229 side-chain, than is the case for an unsubstituted phenyl

Figure 3. Stereo-diagram comparing the NNRTI binding sites of wild-type and Tyr181Cys mutant RTs for the following complexes: (a) nevirapine, (b) TNK-651, (c) PETT-2, and (d) efavirenz. The thinner bonds show the mainchain backbone with wild-type RT coloured as dark grey and the mutant RT as light grey. Side-chains and the inhibitors are shown with thicker bonds with wild-type RT coloured brown and mutant RT as green. For clarity the sidechain of 181 and inhibitor are shown in red for wild-type RT and in cyan for the mutant. The broken yellow lines represent hydrogen bonds.

802

HIV-1 RT Drug Resistance Mutations

Figure 4. Stereo-diagram comparing the NNRTI binding sites of wild-type and Tyr188Cys mutant RTs for the following: (a) unliganded, (b) nevirapine complex and (c) UC-781 complex. The colour scheme for backbone, inhibitors and side-chains is the same as in Figure 3.

group such as for TNK-651.19,28 S-1153 shows 13.5-fold weaker binding to Tyr181Cys RT than to the wild-type,29 and thus the 3,5-substituted phenyl is slightly less effective than the propynyl cyclopropyl group of efavirenz in giving resilience to this RT mutant. A similar example is the

emivirine analogue GCA-186, where addition of 3,5 dimethyl substituents to the phenyl ring gives 20-fold greater resilience to the Tyr181Cys mutation than emivirine itself.30 It is hoped that the rationalisation of mechanisms of resistance at the molecular level for NNRTI

803

HIV-1 RT Drug Resistance Mutations

binding to mutant RTs such as described here will lead to the derivation of some general rules that can be applied in the design of much needed novel inhibitors active against drug-resistant HIV.

Materials and Methods Crystallisation and data collection Expression vectors for HIV-1 RT (HXB-2 isolate) containing either Tyr181Cys or Tyr188Cys mutations were constructed using standard site-directed mutagenesis methods.31 Puri®cation of mutant RTs from recombinant Escherichia coli was based on the ion-exchange procedures previously described.32 All the mutant RT inhibitor complex crystals used in this study were obtained by co-crystallisation and then equilibrated in 50 % (w/v) PEG 3350 prior to data collection.33,34 The unliganded Tyr188Cys crystals were obtained by removal of inhibitor from co-crystals of RT with nevirapine in the same way as described for wild-type RT with HEPT.1 X-ray data were collected at ESRF, Grenoble, France, using the oscillation method and at the Photon Factory Tsukuba, Japan using a Weissenberg camera.35,36 For Tyr181Cys (nevirapine) and Tyr181Cys (TNK-651) data were collected from three positions of a single crystal at 16  C whilst the remaining data sets were collected from single crystals ¯ash-cooled in liquid propane and maintained at 100 K during data collection. Indexing and integration of data images were carried out with DENZO, and data were merged with SCALEPACK.37 Details of the X-ray data statistics are given in Table 1. Structure solution and refinement The molecule orientation and position in the unit cell were determined using rigid-body re®nement with CNS.38 The initial model for each of the current structures was chosen from our previous RT-NNRTI complexes on the basis of closeness of unit cell parameters. The structures were re®ned with CNS38 using positional, simulated annealing and individual B-factor re®nement with bulk solvent correction and anisotropic B-factor scaling. Model rebuilding was done using O on an SGI O2 workstation.39 For the Tyr181Cys (nevirapine), Tyr181Cys (PETT-2), and Tyr188Cys (unliganded) structures positional restraints were applied to all atoms Ê from the Ca atom of residue 188 greater than 25 A throughout the re®nement due to the smaller number of re¯ections. Table 1 gives the re®nement statistics for the seven structures. Structures of wild-type and mutant RTs were overlapped using SHP.40 Figures were produced using BOBSCRIPT,41 a modi®ed version of MOLSCRIPT42 and rendered with Raster3D.43 Data deposition Coordinates and structure factors for all of the HIV-1 RT mutant structures reported here have been deposited in the PDB for immediate release. The codes are as follows: Tyr181Cys RT (nevirapine), 1JLB; Tyr181Cys RT (TNK-651), 1JLA; Tyr181Cys RT (PETT-2), 1JLC; Tyr181Cys RT (efavirenz), 1JKH; Tyr188Cys RT (unliganded), 1JLE; Tyr188Cys RT (nevirapine), 1JLF; Tyr188Cys RT (UC-781), 1JLG.

Acknowledgements We thank the staff of the ESRF, Grenoble, France and the Photon Factory, Tsukuba, Japan. D.I.S. is a member of the TARA project. We thank the UK MRC for long term funding of the RT work with grants to D.K.S. and D.I.S. The Oxford Centre for Molecular Sciences is supported by the Biotechnology and Biological Sciences Research Council, the Medical Research Council and the Engineering and Physical Sciences Research Council.

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Edited by J. Karn (Received 20 April 2001; received in revised form 24 July 2001; accepted 24 July 2001)