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Mutation of Gln125 to Asn selectively abolishes the thymidylate kinase activity of herpes simplex virus type 1 thymidine kinase.
The broad substrate specificity of herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) has provided the basis for selective antiherpetic therapy and, more recently, suicide gene therapy for the treatment of cancer. We have now constructed an HSV-1 TK mutant enzyme, in which an asparagine (N) residue is substituted for glutamine (Q) at position 125, and have evaluated the effect of this amino acid change on enzymatic activity. In marked contrast with wild-type HSV-1 TK, which displays both thymidine kinase and thymidylate kinase activities, the HSV-1 TK(Q125N) mutant was unable to phosphorylate pyrimidine nucleoside monophosphates but retained significant phosphorylation activity for thymidine and a series of antiherpetic pyrimidine and purine nucleoside analogs. The abrogation of HSV-1 TK-associated thymidylate kinase activity resulted in a 100-fold accumulation of the monophosphate form of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) in osteosarcoma cells transfected with the HSV-1 TK(Q125N) gene compared with osteosarcoma cells expressing wild-type HSV-1 TK. BVDU monophosphate accumulation gave rise to a much greater inhibition of cellular thymidylate synthase in HSV-1 TK(Q125N) gene-transfected cells than wild-type HSV-1 TK gene-transfected osteosarcoma tumor cells without significantly changing the cytostatic potency of BVDU for the HSV-1 TK gene-transfected tumor cells. Accordingly, the presence of the Q125N mutation in HSV-1 TK gene-transfected tumor cells was found to result in a multilog decrease in the cytostatic activity of those pyrimidine nucleoside analogs that in their monophosphate form do not have marked affinity for thymidylate synthase [i.e., 1-beta-D-arabinofuranosylthymine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil].
Structure determination of human semaphorin 4D as an example of the use of MAD in non-optimal cases.
Semaphorins are an important class of signalling molecules involved in axon guidance, immune function and angiogenesis. They are characterized by having an extracellular sema domain of about 500 residues. The steps involved in the determination of the structure of human semaphorin 4D are described here as a case study of selenium MAD phasing in a difficult case with low symmetry, moderate diffraction and low selenium content. A particular feature of this study was the large number of diffraction images required to give data of sufficient quality for structure determination and these data are re-analyzed here to investigate the effects of radiation damage on eventual data quality and to suggest strategies for successful MAD phasing in similar difficult cases.
Susceptibility of feline immunodeficiency virus/human immunodeficiency virus type 1 reverse transcriptase chimeras to non-nucleoside RT inhibitors.
To map the determinants of the lack of susceptibility of feline immunodeficiency virus (FIV) reverse transcriptase (RT) to anti human immunodeficiency virus type 1 (HIV-1) non-nucleoside RT inhibitors (NNRTIs), a variety of chimeric HIV-1/FIV RTs were constructed. The majority of chimeric RTs had an affinity (Km) for their natural substrates comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy was decreased. Whereas HIV-1 RT could be made entirely insensitive to NNRTIs by exchanging the amino acid sequence 97 through 205 of FIV RT, none of the reverse FIV/HIV-1 RT chimeras gained susceptibility to NNRTIs. The amino acids that are thought to be involved in NNRTI susceptibility and that are different from those in HIV-1 RT have also been introduced in FIV RT. These mutant RTs gained virtually no susceptibility to efavirenz or capravirine. Vice versa, when these HIV-1-specific amino acids were replaced by their FIV RT counterparts in HIV-1 RT, susceptibility to the NNRTIs was lost. Thus, replacing segments or substituting relevant amino acids in FIV RT by their HIV-1 RT counterparts did not suffice to make FIV RT sensitive toward NNRTIs and was often accompanied by a decrease or even total loss of polymerase activity. It is postulated that, in contrast to the results found for HIV-1/HIV-2 RT chimeras and supported by the crystal structure of HIV-2 RT, there exist significant differences in the structure and/or flexibility of FIV RTs that may prevent NNRTIs from interacting with the FIV RT.
Molecular-dynamics studies of single-stranded hexitol, altritol, mannitol, and ribose nucleic acids (HNA, MNA, ANA, and RNA, resp.) and of the stability of HNA·RNA, ANA·RNA, and MNA·RNA duplexes
The influence of the orientation of a 3'-OH group on the conformation and stability of hexitol oligonucleotides in complexes with RNA and as single strands in aqueous solution was investigated by molecular-dynamics (MD) simulations with AMBER 4.1. The particle mesh Ewald (PME) method was used for the treatment of long-range electrostatic interactions. An equatorial orientation of the 3'-OH group in the single-stranded D-mannitol nucleic acid (MNA) m(GCGTAGCG) and in the complex with the RNA r(CGCAUCGC) has an unfavorable influence on the helical stability. Frequent H-bonds between the 3'-OH group and the O-C(6') of the phosphate backbone of the following nucleotide explain the distorted conformation of the MNA·RNA complex as well as that of the single MNA strand. This is consistent with experimental results that show lowered hybridization potentials for MNA·RNA complexes. An axial orientation of the 3'-OH group in the D-altritol nucleic acid (ANA) a(GCGTAGCG) leads to a stable complex with the complementary RNA r(CGCAUCGC), as well as to a more highly preorganized single-stranded ANA chain. The averaged conformation of the ANA·RNA complex is similar to that of A-RNA, with only minor changes in groove width, helical curvature, and H-bonding pattern. The relative stabilities of ANA·RNA vs. HNA·RNA (HNA = D-hexitol nucleic acid without 3'-OH group) can be explained by differences in restricted movements, H-bonds, and solvation effects.
7-Deazaxanthine, a novel prototype inhibitor of thymidine phosphorylase.
7-Deazaxanthine (7DX) was identified as a novel inhibitor of thymidine (dThd) phosphorylase (TPase). It inhibited the TPase reaction in a concentration-dependent manner. At 1 mM, it almost completely prevented the TPase-catalysed hydrolysis of dThd to thymine. The 50% inhibitory concentration (IC50 of 7DX was 40 microM in the presence of 100 microM of the natural substrate dThd. 7DX is also endowed with a marked inhibitory effect on angiogenesis. It significantly prevents neovascularisation in the chicken chorioallantoic membrane during development. 7DX is the first purine derivative shown to be a potent inhibitor of purified TPase and angiogenesis.
Crystals of HIV-1 reverse transcriptase diffracting to 2.2 A resolution.
Reverse transcriptase (RT) from the human immunodeficiency virus type 1 has been crystallized in four closely related forms, the best of which diffract X-rays to 2.2 A resolution. The RT was crystallized as a complex with a non-nucleoside inhibitor, either nevirapine or a nevirapine analogue. Crystals grew from 6% PEG 3400 buffered at pH 5. These were of space group P2(1)2(1)2(1) with unit cell parameters a = 147 A, b = 112 A, c = 79 A (form A), with one RT heterodimer in the asymmetric unit. Changes in unit cell parameters and degree of crystalline order were observed on soaking pregrown crystals in various solutions, giving three further sets of unit cells. These were a = 143 A, b = 112, A, c = 79 A (form B), a = 141 A, b = 111 A, c = 73 A (form C), a = 143 A, b = 117 A, c = 66.5 A (form D). The last two forms diffract X-rays to 2.2 A resolution. Structure determinations of these latter crystal forms of RT should give a detailed atomic model for this therapeutically important drug target.
Mutations in the non-nucleoside binding-pocket interfere with the multi-nucleoside resistance phenotype.
OBJECTIVES:To investigate the genotypic and phenotypic effects of in vitro resistance selection with lamivudine and/or the second generation non-nucleoside reverse transcriptase inhibitor (NNRTI) quinoxaline HBY097 using HIV-1 isolates carrying the multi-nucleoside resistance pattern linked to the Q151M mutation. METHODS:Virus strains were selected in C8166 cells in the presence of increasing concentrations of lamivudine or HBY097. In parallel control experiments, the virus was cultured in C8166 cells in the absence of drugs. The entire reverse transcriptase encoding region was amplified using polymerase chain reaction and was subsequently sequenced. Antiviral activities of drugs were evaluated in C8166 cells. RESULTS:High-level resistant viruses were selected rapidly in the presence of lamivudine and quinoxaline (less than 10 passages). The multi-nucleoside resistance mutations were stable during in vitro resistance selection. Lamivudine elicited the acquisition of the M184I mutation. Phenotypic resistance to all nucleoside-analog reverse transcriptase inhibitors (NRTIs) was increased when M184I was added to the multi-nucleoside resistance background in the absence of NNRTI-resistance mutations. In most cases of HBY097 resistance selection, at least two mutations associated with NNRTI resistance resulted in high-level NNRTI resistance. The NNRTI resistance-related mutations partially reversed the phenotypic resistance to most NRTIs, except to abacavir. The addition of the M184I mutation to the NNRTI-multi-nucleoside resistance set abolished this antagonizing effect for didanosine, zalcitabine and lamivudine, but further potentiated the phenotypic reversal for zidovudine and stavudine. CONCLUSION:Changes in the non-nucleoside binding pocket must affect the conformation of residues at the dNTP binding site, and can result in a partial phenotypic reversal of the multi-nucleoside resistance phenotype.
Phenylethylthiazolylthiourea (PETT) non-nucleoside inhibitors of HIV-1 and HIV-2 reverse transcriptases. Structural and biochemical analyses.
Most non-nucleoside reverse transcriptase (RT) inhibitors are specific for HIV-1 RT and demonstrate minimal inhibition of HIV-2 RT. However, we report that members of the phenylethylthiazolylthiourea (PETT) series of non-nucleoside reverse transcriptase inhibitors showing high potency against HIV-1 RT have varying abilities to inhibit HIV-2 RT. Thus, PETT-1 inhibits HIV-1 RT with an IC(50) of 6 nM but shows only weak inhibition of HIV-2 RT, whereas PETT-2 retains similar potency against HIV-1 RT (IC(50) of 5 nM) and also inhibits HIV-2 RT (IC(50) of 2.2 microM). X-ray crystallographic structure determinations of PETT-1 and PETT-2 in complexes with HIV-1 RT reveal the compounds bind in an overall similar conformation albeit with some differences in their interactions with the protein. To investigate whether PETT-2 could be acting at a different site on HIV-2 RT (e.g. the dNTP or template primer binding site), we compared modes of inhibition for PETT-2 against HIV-1 and HIV-2 RT. PETT-2 was a noncompetitive inhibitor with respect to the dGTP substrate for both HIV-1 and HIV-2 RTs. PETT-2 was also a noncompetitive inhibitor with respect to a poly(rC).(dG) template primer for HIV-2 RT. These results are consistent with PETT-2 binding in corresponding pockets in both HIV-1 and HIV-2 RT with amino acid sequence differences in HIV-2 RT affecting the binding of PETT-2 compared with PETT-1.
Crystallographic analysis of the binding modes of thiazoloisoindolinone non-nucleoside inhibitors to HIV-1 reverse transcriptase and comparison with modeling studies.
We have determined the crystal structures of thiazoloisoindolinone non-nucleoside inhibitors in complex with HIV-1 reverse transcriptase to high-resolution limits of 2.7 A (BM +21.1326) and 2. 52 A (BM +50.0934). We find that the binding modes of this series of inhibitors closely resemble that of "two-ring" non-nucleoside reverse transcriptase inhibitors. The structures allow rationalization of stereochemical requirements, structure-activity data, and drug resistance data. Comparisons with our previous structures suggest modifications to the inhibitors that might improve resilience to drug-resistant mutant forms of reverse transcriptase. Comparison with earlier modeling studies reveals that the predicted overlap of thiazoloisoindolinones with TIBO was largely correct, while that with nevirapine was significantly different.
Poly(2-acrylamido-2-methyl-1-propanamide) (PAMPA): A neutral, water-soluble synthetic polymer with double-stranded helix conformation
A new synthetic polymer, poly(2-acrylamido-2-methyl-1-propanamide) (PAMPA), is described. It combines the structural features of poly(leucine) and poly(glutamine), its molecular weight averages 42000 D, and it contains ca. 270 residues. A 13C-NMR-tacticity study indicates that it consists of a mixture of syndiotactic (38%), heterotactic (48%), and isotactic (17%) polymer. PAMPA has a tendency to self-organize into a coiled-coil double-stranded helix structure in aqueous solution, as determined by Fourier-transform infrared spectroscopy (FT-IR). Molecular modeling of PAMPA shows that the helix formation is driven by repeated intramolecular H-bonds between nearest-neighbor amide groups, and that the structure is further stabilized by hydrophobic interactions between the two lateral Me groups. PAMPA has a neutral structure, is highly water-soluble, and demonstrates temperature stability.
Characterization of multiple nuclear localization signals in herpes simplex virus type 1 thymidine kinase.
We have reported previously that the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) fused with green fluorescent protein (GFP) is localized in the nucleus of HSV-1 TK-GFP gene-transfected cells (Degrève et al. (1998) J. Virol. 72, 9535-9543). Deletion of the N-terminal 34 amino acids or selective mutation of the nonapeptide (25)RRTALRPRR(33), located in the N-terminal region of HSV-1 TK, resulted in the loss of the specific nuclear localization of HSV-1 TK. Utilizing information on the crystallographic structure of HSV-1 TK, we have now identified three additional putative nuclear localization signals and evaluated their potential role in the nuclear trafficking of HSV-1 TK by site-directed mutagenesis. We found that the sites containing the amino acids R236-R237 and K317-R318 are absolutely required for specific nuclear targeting of HSV-1 TK. The K317-R318 region, located at the interface between the two monomers in the dimeric HSV-1 TK structure, could act as a nuclear localization signal for monomeric HSV-1 TK. Alternatively, crystallographic data indicate that R318 might be essential for the formation of the TK dimer, and therefore it is required if HSV-1 TK is transported as a dimer.
The A167Y mutation converts the herpes simplex virus type 1 thymidine kinase into a guanosine analogue kinase.
The thymidine (dThd) kinase (TK) encoded by herpes simplex virus type 1 (HSV-1) is not only endowed with dThd kinase, but also with thymidylate (dTMP) kinase and 2'-deoxycytidine (dCyd) kinase (dCK) activity. HSV-1 TK also recognizes a variety of antiherpetic guanine nucleoside analogues such as acyclovir (ACV), ganciclovir (GCV), lobucavir (LBV), penciclovir (PCV), and others (i.e., A5021). Site-directed mutagenesis of the highly conserved Ala-167 to Tyr in HSV-1 TK completely abolished TK, dTMP-K, and dCK activity, but maintained ACV-, GCV-, LBV-, PCV-, and A5021-phosphorylating capacity. A variety of 5-substituted pyrimidine nucleoside substrates, but also a number of selective HSV-1 TK inhibitors structurally related to thymine lost significant binding affinity for the mutant enzyme and did not markedly compete with GCV phosphorylation by the mutant enzyme. These findings could be explained by computer-assisted modeling data that revealed steric hindrance of the pyrimidine ring in the HSV-1 TK active site by the large 4-hydroxybenzyl ring of 167-Tyr, while the positioning of the purine ring of guanine-based HIV-1 TK substrates in the active site was kept virtually unaltered. Surprisingly, the efficiency of conversion the antiherpetic 2'-deoxyguanosine analogues ACV, GCV, LBV, PCV, and A5021 to their phosphorylated forms by the A167Y mutant HSV-1 TK was far more pronounced than for the wild-type enzyme. Therefore, the single A167Y mutation converts the wild-type HSV-1 TK from a predominantly pyrimidine nucleos(t)ide kinase into a virtually exclusive purine (guanine) nucleoside analogue kinase.
Kinetic analysis of novel multisubstrate analogue inhibitors of thymidine phosphorylase.
A kinetic analysis was performed for the novel 1-(8-phosphonooctyl)-6-amino-5-bromouracil and 1-(8-phosphonooctyl)-7-deazaxanthine inhibitors of Escherichia coli thymidine (dThd) phosphorylase (TPase). The structure of the compounds was rationally designed based on the available crystal structure coordinates of bacterial TPase. These inhibitors reversibly inhibited TPase. Kinetic analysis revealed that the compounds inhibited TPase in a purely competitive or mixed fashion not only when dThd, but also when inorganic phosphate (Pi), was used as the variable substrate. In contrast, the free bases 6-amino-5-bromouracil and 7-deazaxanthine behaved as non-competitive inhibitors of the enzyme in the presence of variable Pi concentrations while being competitive or mixed with respect to thymine as the natural substrate. Our kinetic data thus revealed that the novel 1-(8-phosphonooctyl)pyrimidine/purine derivatives are able to function as multisubstrate inhibitors of TPase, interfering at two different sites (dThd(Thy)- and phosphate-binding site) of the enzyme. To our knowledge, the described compounds represent the first type of such multisubstrate analogue inhibitors of TPase; they should be considered as lead compounds for the development of mechanistically novel type of TPase inhibitors.
Interaction of cycloSal-pronucleotides with cholinesterases from different origins. A structure-activity relationship.
A large number of cycloSal-nucleotide triesters 1-49 have been studied concerning their ability to inhibit cholinesterases of different origins as well as to inhibit HIV replication in cell culture. It was shown that none of the triesters showed inhibitory effects against human acetylcholinesterase (AChE; isolated enzyme) as well as against AChE from beef erythrocytes and calf serum. In contrast, inhibition of butyrylcholinesterase (BChE) has been observed for some triesters in human and mouse serum. cycloSal pronucleotides showed strong competitive inhibition with respect to the substrate acetylcholine chloride (K(i)/K(m): approximately 2 x 10(-5)) and acted by time-dependent irreversible inhibition of the human serum BChE. Detailed studies demonstrated that the inhibitory effect against BChE is dependent on the nucleoside analogue, the substitution pattern of the cycloSal-moiety, and particularly on the stereochemistry at the phosphorus atom. Structural requirements to avoid the inhibition of BChE by cycloSal-nucleotide triesters have been elucidated in the reported study.
The crystal structure of UMP kinase from Bacillus anthracis (BA1797) reveals an allosteric nucleotide-binding site.
Uridine monophosphate (UMP) kinase is a conserved enzyme that catalyzes the ATP-driven conversion of uridylate monophosphate into uridylate diphosphate, an essential metabolic step. In prokaryotes, the enzyme exists as a homohexamer that is regulated by various metabolites. Whereas the enzymatic mechanism of UMP kinase (UK) is well-characterized, the molecular basis of its regulation remains poorly understood. Here we report the crystal structure of UK from Bacillus anthracis (BA1797) in complex with ATP at 2.82 A resolution. It reveals that the cofactor, in addition to binding in the active sites, also interacts with separate binding pockets located near the center of the hexameric structure. The existence of such an allosteric binding site had been predicted by biochemical studies, but it was not identified in previous crystal structures of prokaryotic UKs. We show that this putative allosteric pocket is conserved across different bacterial species, suggesting that it is a feature common to bacterial UKs, and we present a structural model for the allosteric regulation of this enzyme.
An ion-channel modulator from the saliva of the brown ear tick has a highly modified Kunitz/BPTI structure.
Ra-KLP, a 75 amino acid protein secreted by the salivary gland of the brown ear tick Rhipicephalus appendiculatus has a sequence resembling those of Kunitz/BPTI proteins. We report the detection, purification and characterization of the function of Ra-KLP. In addition, determination of the three-dimensional crystal structure of Ra-KLP at 1.6 A resolution using sulphur single-wavelength anomalous dispersion reveals that much of the loop structure of classical Kunitz domains, including the protruding protease-binding loop, has been replaced by beta-strands. Even more unusually, the N-terminal portion of the polypeptide chain is pinned to the "Kunitz head" by two disulphide bridges not found in classical Kunitz/BPTI proteins. The disulphide bond pattern has been further altered by the loss of the bridge that normally stabilizes the protease-binding loop. Consistent with the conversion of this loop into a beta-strand, Ra-KLP shows no significant anti-protease activity; however, it activates maxiK channels in an in vitro system, suggesting a potential mechanism for regulating host blood supply during feeding.
Models which explain the inhibition of reverse transcriptase by HIV-1-specific (thio)carboxanilide derivatives.
The (thio)carboxanilide derivatives are potent and selective inhibitors of HIV-1 reverse transcriptase (RT) and have a favourable antiviral activity spectrum. To understand better their mode of action, and to provide a structural basis for further improvement, models of RT complexed with four (thio)carboxanilide inhibitors (UC781, UC10, UC38 and UC84) have been constructed based on the X-ray structure of RT complexed with 9-chloro-TIBO. In the models, the protein conformation is similar to that of the RT-TIBO complex and the complexes are stabilised by hydrogen bonding between the inhibitors and the main chain oxygen of Lys101. Significant hydrophobic interactions include those with Leu100, Val106, Val179, Tyr188, Phe227, Leu234, and His235. The thiocarboxanilides UC781 and UC10 also make important hydrophobic interactions with Trp229. The models are consistent with the inhibitors' relative antiviral potencies and the observed resistance data. They further predict that mutations to Phe227, Trp229, or Leu234 might confer resistance. Since these are not observed, some constraining structural or functional role for these residues in the active enzyme is suggested.
Mutational analysis of Tyr-318 within the non-nucleoside reverse transcriptase inhibitor binding pocket of human immunodeficiency virus type I reverse transcriptase.
The highly conserved Tyr-318 is part of the non-nucleoside reverse transcriptase inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type I reverse transcriptase (RT) and makes contact within 4 A with the NNRTIs in all reported RT/NNRTI complexes. Using site-directed mutagenesis, six mutant RTs were constructed bearing the mutations Y318H, Y318K, Y318L, Y318C, Y318W, and Y318F. We found that only the Y318W and Y318F mutant RTs retained substantial RT activity, whereas the catalytic activities of the Y318K, Y318C, Y318H, and Y318L RT mutants were less than 5% of the wild-type activity. The Y318F mutant RT retained substantial sensitivity to the majority of NNRTIs tested, whereas the Y318W mutant RT showed varying degrees of resistance to NNRTIs. Subunit-specific site-directed mutagenesis revealed that there was no difference in the catalytic activity or resistance/sensitivity spectrum toward NNRTIs regardless of whether the Tyr-318 mutation was introduced in both subunits or only in the p66 subunit of RT. Recombinant viruses harboring the Y318F or Y318W mutation in the RT showed a similar resistance/sensitivity pattern to NNRTIs as their corresponding 318 mutant recombinant RTs. Our findings stress a functional or structural role for Tyr-318 in wild-type RT and argue for the design of novel NNRTIs that interact more closely with this amino acid in the NNRTI-specific pocket of human immunodeficiency virus type I RT.
Structural and kinetic basis for heightened immunogenicity of T cell vaccines.
Analogue peptides with enhanced binding affinity to major histocompatibility class (MHC) I molecules are currently being used in cancer patients to elicit stronger T cell responses. However, it remains unclear as to how alterations of anchor residues may affect T cell receptor (TCR) recognition. We correlate functional, thermodynamic, and structural parameters of TCR-peptide-MHC binding and demonstrate the effect of anchor residue modifications of the human histocompatibility leukocyte antigens (HLA)-A2 tumor epitope NY-ESO-1(157-165)-SLLMWITQC on TCR recognition. The crystal structure of the wild-type peptide complexed with a specific TCR shows that TCR binding centers on two prominent, sequential, peptide sidechains, methionine-tryptophan. Cysteine-to-valine substitution at peptide position 9, while optimizing peptide binding to the MHC, repositions the peptide main chain and generates subtly enhanced interactions between the analogue peptide and the TCR. Binding analyses confirm tighter binding of the analogue peptide to HLA-A2 and improved soluble TCR binding. Recognition of analogue peptide stimulates faster polarization of lytic granules to the immunological synapse, reduces dependence on CD8 binding, and induces greater numbers of cross-reactive cytotoxic T lymphocyte to SLLMWITQC. These results provide important insights into heightened immunogenicity of analogue peptides and highlight the importance of incorporating structural data into the process of rational optimization of superagonist peptides for clinical trials.
The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design.
BACKGROUND: HIV reverse transcriptase (RT) is a key target of anti-AIDS therapies. Structural studies of HIV-1 RT, unliganded and complexed with different non-nucleoside inhibitors (NNIs), have pointed to a common mode of binding and inactivation through distortion of the polymerase catalytic site by NNIs containing two hinged rings. The mode of binding of the TIBO family of inhibitors is of interest because these compounds do not fit the two-hinged-ring model. RESULTS: The structure of HIV-1 RT complexed with 9-chloro-TIBO (R82913) has been determined at 2.6 A resolution. As reported for the lower resolution analysis of another TIBO compound, this inhibitor binds at the same site as other NNIs, but our higher resolution study reveals the Cl-TIBO is distorted from the conformation seen in crystals of the inhibitor alone. This allows Cl-TIBO to mimic the binding of NNIs containing two hinged rings. Inhibitor-protein interactions are again predominantly hydrophobic and the protein conformation corresponds to that seen in complexes with other tight-binding NNIs. CONCLUSIONS: Although Cl-TIBO is chemically very different from other NNIs, it achieves remarkable spatial equivalence and shape complementarity with other NNIs on binding to RT. Comparison of the different RT-NNI complexes suggests modifications to the TIBO group of inhibitors which might enhance their binding and hence, potentially, their therapeutic efficacy.