Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD 20892-0520.
Imaging Sciences Laboratory, Center for Information Technology, NIH, Bethesda, MD 20892-5624.
Proc Natl Acad Sci U S A. 2019 Sep 3;116(36):17809-17816. doi: 10.1073/pnas.1911086116. Epub 2019 Aug 5.
HIV type I (HIV-1) reverse transcriptase (RT) catalyzes the conversion of viral RNA into DNA, initiating the chain of events leading to integration of proviral DNA into the host genome. RT is expressed as a single polypeptide chain within the Gag-Pol polyprotein, and either prior to or following excision by HIV-1 protease forms a 66 kDa chain (p66) homodimer precursor. Further proteolytic attack by HIV-1 protease cleaves the ribonuclease H (RNase H) domain of a single subunit to yield the mature p66/p51 heterodimer. Here, we probe the spatial domain organization within the p66 homodimer using pulsed Q-band double electron-electron resonance (DEER) EPR spectroscopy to measure a large number of intra- and intersubunit distances between spin labels attached to surface-engineered cysteines. The DEER-derived distances are fully consistent with the structural subunit asymmetry found in the mature p66/p51 heterodimer in which catalytic activity resides in the p66 subunit, while the p51 subunit purely serves as a structural scaffold. Furthermore, the p66 homodimer precursor undergoes a conformational change involving the thumb, palm, and finger domains in one of the subunits (corresponding to the p66 subunit in the mature p66/p51 heterodimer) from a closed to a partially open state upon addition of a nonnucleoside inhibitor. The relative orientation of the domains was modeled by simulated annealing driven by the DEER-derived distances. Finally, the RNase H domain that is cleaved to generate p51 in the mature p66/p51 heterodimer is present in 2 major conformers. One conformer is fully solvent accessible thereby accounting for the observation that only a single subunit of the p66 homodimer precursor is susceptible to HIV-1 protease.
HIV 型 1（HIV-1）逆转录酶（RT）催化病毒 RNA 转化为 DNA，启动导致前病毒 DNA 整合到宿主基因组的事件链。RT 作为 Gag-Pol 多蛋白中的单个多肽链表达，并且在 HIV-1 蛋白酶切割之前或之后形成 66 kDa 链（p66）同源二聚体前体。HIV-1 蛋白酶的进一步蛋白水解攻击将单个亚基的核糖核酸酶 H（RNase H）结构域切割，产生成熟的 p66/p51 异源二聚体。在这里，我们使用脉冲 Q 带双电子-电子共振（DEER）EPR 光谱研究 p66 同源二聚体中的空间结构域组织，以测量附着在表面工程半胱氨酸上的自旋标记之间的大量亚基内和亚基间距离。DEER 衍生的距离与成熟的 p66/p51 异源二聚体中发现的结构亚基不对称性完全一致，其中催化活性位于 p66 亚基中，而 p51 亚基纯粹作为结构支架。此外，p66 同源二聚体前体经历构象变化，涉及一个亚基（对应于成熟的 p66/p51 异源二聚体中的 p66 亚基）的拇指、手掌和手指结构域，从封闭状态转变为部分开放状态添加非核苷抑制剂后。通过由 DEER 衍生的距离驱动的模拟退火对结构域的相对取向进行建模。最后，在成熟的 p66/p51 异源二聚体中切割产生 p51 的 RNase H 结构域存在 2 种主要构象。一种构象完全可溶，从而解释了仅 p66 同源二聚体前体的单个亚基对 HIV-1 蛋白酶敏感的观察结果。
