Crystal Structures of 8-CI and 9-CI TIBO Complexed with Wild-type HIV-1 RT and 8-CI TIBO Complexed with the Tyr181 Cys HIV-1 RT Drug-resistant Mutant

摘要

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is an important target for chemotherapeutic agents used in the treatment of AIDS; the TIBO compounds are potent non-nucleoside inhibitors of HIV-1 RT (NNRTIs). Crystal structures of HIV-1 RT coinplexed with 8-CI TIBO (R86183,IC_(50) = 4.6 nM) and 9-C1 TIBO (R82913, IC_(50) = 33 nM) have been determined at 3.0 A resolution. Mutant HIV-1 RT, containing Cys in place of Tyr at position 181 (TyrlSlCys), is highly resistant to many NNRTIs and HIV-1 variants containing this mutation have been selected in both cell culture and clinical trials. We also report the crystal structure of Tyrl8lCys HIV-1 RT in complex with 8-C1 TIBO (IC_(50) = 130 nM) determined at 3.2 A resolution. Averagingof the electron density maps computed for different HIV-1 RT/NNRTI complexes and from diffraction datasets obtained using a synchrotron source from frozen (-165 °C) and cooled (-10 °C) crystals of the same complex was employed to improve the quality ofelectron density maps and to reduce model bias. The overall locations and conformations of the bound inhibitors in the complexes containing wild-type HIV-1 RT and the two TIBO inhibitors are very similar, as are the overall shapes and volumes of the non-nucleoside inhibitor-binding pocket (NNIBP). The major differences between the two wild-type HIV-1 RT/TIBO complexes occur in the vicinity of the TIBO chlorine substituents and involve the polypeptide segments around the beta5-beta6 connecting loop (residues 95 to 105) and the [beta13-beta14 hairpin (residues 235 and 236). In all known structures of HIV-1 RT/NNRTI complexes, including these two, the position of the (beta12-beta13 hairpin or the "primer grip" is significantly displaced relative to the position in the structure of HIV-1 RT coinplexed with a double-stranded DNA and in unliganded HIV-1 RT structures. Since the primer grip helps to position the template-primer, this displacement suggests that binding of NNRTIs would affect the relative positions of the primer terminus and the polymerase active site. This could explain biochemical data showing that NNRTI binding to HIV-1 RT reduces efficiency of the chemical step of DNA polymerization, but does not prevent binding of either dNTPs or DNA.When the structure of the Tyrl8lCys mutant HIV-1 RT in complex with 8-C1 TIBO is compared with the corresponding structure containing wild-type HIV-1 RT, the overall conformations of Tyrl8lCys and wild-type HIV-1 RT and of the 8-C1 TIBO inhibitors are very similar. Some positional changes in the polypeptide backbone of the beta6-betal0-beta9 sheet containing residue 181 are observed when the Tyrl8lCys and wild-type complexes are compared, particularly near residue Vall79 of beta9 In the beta51 subunit,the Cysl8l side-chain is oriented in a similar direction to the Tyrl8l side-chain in the wild -type complex. However, the electron density corresponding to the sulfur of the Cysl8l side-chain in the p66 subunit is very weak, indicating that the thiol group is disordered, presumably because there is no significant interaction with either 8-C1 TIBO or nearby amino acid residues In the mutant complex, there are slight "rearrangements of the side-chains of other amino acid residues in the NNIBP and of the flexible dimethylallyl group of 8-C1 TIBO; these conformational changes could potentially compensate for the interactions that were lost when the relatively large tyrosine at position 181 was replaced by a less bulky cysteine residue. In the correspondingwild-type complex, TyrlSl in the p66 subunit has significant interactions with the bound inhibitor and the position of the Tyrl8l side chain is well defined in both subunits. Apparently the Tytl81->Cys mutation eliminates favorable contacts of the aromatic ring of the tyrosine and the bound inhibitor, reducing the stability of NNRTI binding. This is consistent with the observation that the Tyrl8lCys mutant HIV-1 RT is more resistant to NNRTTs that ha