Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function
Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function
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DOI：
10.1016/s0969-2126(99)80172-5
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发表时间：
1999-09-15
期刊：
影响因子：
5.7
通讯作者：
Torchia, DA
中科院分区：
文献类型：
--
作者：
Ishima, R;Freedberg, DI;Torchia, DA
背景：全氘化蛋白质的 H-1 和 N-15 横向弛豫测量非常适合检测毫秒-微秒时间尺度上的主链构象波动。通过测量 H-1 和 N-15 的弛豫来识别该时间尺度上的构象交换，对于阐明蛋白质中功能相关的构象变化具有很大的希望。结果：我们测量了游离形式和抑制剂结合形式的 HIV-1 蛋白酶主链酰胺的横向 H-1 和 N-15 弛豫率。对这些速率的分析，作为有效旋转框架场的函数获得，提供了有关蛋白质几个区域结构波动的时间尺度的信息。覆盖抑制剂结合蛋白活性位点的瓣在游离蛋白中在 100 μs 时间尺度上经历主链 (phi、psi) 角度的显着变化。此外，结合形式的单体间β-片层界面（从蛋白酶结构研究看来是刚性的）被发现在毫秒时间尺度上波动。 结论：我们提出了游离 HIV-1 蛋白酶中瓣打开机制的工作模型，该模型涉及从半开放构象到开放构象的转变，这是通过 Phe53 环与底物的相互作用促进的。我们还发现了β-折叠单体间界面的惊人波动，这表明蛋白酶成熟的结构要求。因此，通过 H-1 和 N-15 横向弛豫测量确定的缓慢构象波动可能与蛋白质的生物学功能有关。
Background: H-1 and N-15 transverse relaxation measurements on perdeuterated proteins are ideally suited for detecting backbone conformational fluctuations on the millisecond-microsecond timescale. The identification of conformational exchange on this timescale by measuring the relaxation of both H-1 and N-15 holds great promise for the elucidation of functionally relevant conformational changes in proteins.Results: We measured the transverse H-1 and N-15 relaxation rates of backbone amides of HIV-1 protease in its free and inhibitor-bound forms. An analysis of these rates, obtained as a function of the effective rotating frame field, provided information about the timescale of structural fluctuations in several regions of the protein. The flaps that cover the active site of the inhibitor-bound protein undergo significant changes of backbone (phi, psi) angles, on the 100 mu s timescale, in the free protein. In addition, the intermonomer beta-sheet interface of the bound form, which from protease structure studies appears to be rigid, was found to fluctuate on the millisecond timescale.Conclusions: We present a working model of the flap-opening mechanism in free HIV-1 protease which involves a transition from a semi-open to an open conformation that is facilitated by interaction of the Phe53 ring with the substrate. We also identify a surprising fluctuation of the beta-sheet intermonomer interface that suggests a structural requirement for maturation of the protease. Thus, slow conformational fluctuations identified by H-1 and N-15 transverse relaxation measurements can be related to the biological functions of proteins.