Ren J, Nichols C, Bird L, Chamberlain P, Weaver K, Short S, Stuart D I, Stammers D K
Structural Biology Division, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
J Mol Biol. 2001 Sep 28;312(4):795-805. doi: 10.1006/jmbi.2001.4988.
Mutations at either Tyr181 or Tyr188 within HIV-1 reverse transcriptase (RT) give high level resistance to many first 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 first and second generation NNRTIs as well as one example of an unliganded mutant RT. These are Tyr181Cys RT (TNK-651) to 2.4 A, Tyr181Cys RT (efavirenz) to 2.6 A, Tyr181Cys RT (nevirapine) to 3.0 A, Tyr181Cys RT (PETT-2) to 3.0 A, Tyr188Cys RT (nevirapine) to 2.6 A, Tyr188Cys RT (UC-781) to 2.6 A and Tyr188Cys RT (unliganded) to 2.8 A resolution. In the two previously 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 first 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 first generation compounds, providing a simple explanation for the resilience of second generation NNRTIs, as such interactions make much less significant contribution to their binding.
HIV-1逆转录酶（RT）中Tyr181或Tyr188位点的突变会使病毒对许多第一代非核苷类逆转录酶抑制剂（NNRTIs）产生高水平耐药性，比如抗艾滋病药物奈韦拉平。相比之下，第二代抑制剂，例如依法韦仑，对这些突变表现出更强的耐受性。为了理解这些差异的结构基础，我们测定了一系列七个突变RT与第一代和第二代NNRTIs复合物的晶体结构，以及一个未结合配体的突变RT的晶体结构。这些结构分别是Tyr181Cys RT（TNK-651），分辨率为2.4 Å；Tyr181Cys RT（依法韦仑），分辨率为2.6 Å；Tyr181Cys RT（奈韦拉平），分辨率为3.0 Å；Tyr181Cys RT（PETT-2），分辨率为3.0 Å；Tyr188Cys RT（奈韦拉平），分辨率为2.6 Å；Tyr188Cys RT（UC-781），分辨率为2.6 Å；以及Tyr188Cys RT（未结合配体），分辨率为2.8 Å。在之前发表的两个HIV-1逆转录酶181或188位点发生突变的结构中，在p66亚基（包含抑制剂结合口袋）内未观察到突变残基的侧链电子密度。相比之下，在这里报道的所有七个结构中，突变侧链都可以在NNRTI口袋中看到，排除了无序状态导致耐药机制的可能性。对于第二代化合物依法韦仑与Tyr181Cys RT以及UC-781与Tyr188Cys RT，与野生型RT相比，结合位点内的抑制剂只有小的重排，第一代化合物TNK-651、PETT-2和奈韦拉平与Tyr181Cys RT的情况也是如此。然而，对于奈韦拉平与Tyr188Cys RT，药物分子有更显著的移动。我们得出结论，蛋白质构象变化和药物分子在突变位点内的重排不是这些特定抑制剂/突变体组合的普遍特征。Tyr181Cys和Tyr188Cys RT突变导致耐药性的主要原因是第一代化合物失去了芳香环堆积相互作用，这为第二代NNRTIs的耐受性提供了一个简单的解释，因为这种相互作用对它们结合的贡献要小得多。
