Drug resistance is a serious problem for controlling the HIV/AIDS pandemic. Current antiviral drugs show several orders of magnitude worse inhibition of highly resistant clinical variant PRS17 of HIV-1 protease compared with wild-type protease. We have analyzed the effects of a common resistance mutation G48V in the flexible flaps of the protease by assessing the revertant PRS17_V48G for changes in enzyme kinetics, inhibition, structure, and dynamics. Both PRS17 and the revertant showed about 10-fold poorer catalytic efficiency than wild-type enzyme (0.55 and 0.39 μM⁻¹min⁻¹ compared to 6.3 μM⁻¹min⁻¹). Clinical inhibitors, amprenavir and darunavir, showed 2-fold and 8-fold better inhibition, respectively, of the revertant than of PRS17, although the inhibition constants for PRS17_V48G were still 25 to 1,200-fold worse than for wild-type protease. Crystal structures of inhibitor-free revertant and amprenavir complexes with revertant and PRS17 were solved at 1.3–1.5 Å resolution. The amprenavir complexes of PRS17_V48G and PRS17 showed no significant differences in the interactions with inhibitor, although changes were observed in the conformation of Phe53 and the interactions of the flaps. The inhibitor-free structure of the revertant showed flaps in an open conformation, however, the flap tips do not have the unusual curled conformation seen in inhibitor-free PRS17. Molecular dynamics simulations were run for 1 μs on the two inhibitor-free mutants and wild-type protease. PRS17 exhibited higher conformational fluctuations than the revertant, while the wild-type protease adopted the closed conformation and showed the least variation. The second half of the simulations captured the transition of the flaps of PRS17 from a closed to a semi-open state, whereas the flaps of PRS17_V48G tucked into the active site and the wild-type protease retained the closed conformation. These results suggest that mutation G48V contributes to drug resistance by altering the conformational dynamics of the flaps.

耐药性是控制艾滋病毒/艾滋病流行的一个严重问题。与野生型蛋白酶相比，目前的抗病毒药物对 HIV-1 蛋白酶的高度耐药临床变异 PRS17 的抑制效果要差几个数量级。我们通过评估回复型 PRS17 _V48G在酶动力学、抑制、结构和动力学方面的变化，分析了蛋白酶柔性瓣中常见的抗性突变 G48V 的影响。PRS17 和回复体均显示出比野生型酶低约 10 倍的催化效率（0.55 和 0.39 μM ^-1 min ^-1相比 6.3 μM ^-1 min ^-1）。_{尽管 PRS17 V48G}的抑制常数仍比野生型蛋白酶低 25 至 1,200 倍，但临床抑制剂安普那韦和地瑞那韦对回复体的抑制作用分别是 PRS17 的 2 倍和 8 倍。以 1.3–1.5 Å 分辨率解析无抑制剂回复体和安普那韦复合体与回复体和 PRS17 的晶体结构。PRS17 _{V48G的安普那韦复合物}和 PRS17 在与抑制剂的相互作用方面没有显着差异，尽管在 Phe53 的构象和皮瓣的相互作用中观察到了变化。回复体的无抑制剂结构显示皮瓣呈开放构象，然而，皮瓣尖端没有在不含抑制剂的 PRS17 中看到的不寻常的卷曲构象。对两种不含抑制剂的突变体和野生型蛋白酶进行 1 μs 的分子动力学模拟。PRS17的构象波动高于回复体，而野生型蛋白酶采用闭合构象，变异最小。模拟的后半部分捕捉到 PRS17 的襟翼从关闭状态到半打开状态的过渡，而 PRS17 _{V48G的襟翼}塞入活性位点，野生型蛋白酶保持闭合构象。这些结果表明突变 G48V 通过改变皮瓣的构象动力学有助于耐药性。