Combining Molecular Simulations and Biophysical Methods to Characterize Conformational Dynamics of the HIV-1 Envelope Glycoprotein

项目总结 HIV-1包膜糖蛋白刺突蛋白(Env)介导病毒进入靶细胞。因为Env是唯一 病毒粒子表面的病毒蛋白，它是开发潜在疫苗和小分子的核心 进入抑制药。Env是一种独特的灵活的分子机器，并对其免疫原性有深刻的理解 而对抑制的敏感性需要对其原子解析的构象动力学的评价。 使用截断的、增溶的和稳定的环境结构的结构研究已经产生了详细的原子 其主要开放和闭合构象状态的模型。全长围护结构研究的新进展 支持将不对称闭合构象识别为“默认中间状态”(DIS)，揭示了 第四纪不对称性在环境构象动力学中的潜在关键作用的细节。同时， Env的单分子FRET(SmFRET)和交联质谱(XL-MS)都表明 存在至少一种构象状态，有时是占优势的构象状态，这种状态在结构上没有 特色化的。然而，这种“State-1”构象似乎与免疫识别和 对小分子抑制剂的敏感性。我们将利用先进的分子动力学(MD)方法 包括靶向MD、温度加速MD和弦方法，以提供原子级模型 HIV-1env从闭合(状态2)到开放(状态3)构象状态的开放并识别关键 将State-2与鲜为人知的State-1 Env分开的结构变化。MD模拟方法WE USE将结合来自多角度smFRET和XL-MS的偏向，它们将反过来提供方向 为了扩展这些实验中使用的Env构造集，建立了一种迭代方法，该方法 逐步更好地定义过渡机制和状态1。对艾滋病毒-1环境的原子水平理解 构象动力学，识别尚未被结构表征的预触发构象 国家，以及环境病毒激活融合的机制将为免疫原设计和抗病毒提供信息 治疗。

PROJECT SUMMARY The HIV-1 envelope glycoprotein spike (Env) mediates viral entry into target cells. Because Env is the only viral protein on the virion surface, it is central to the development of potential vaccines and small-molecule entry inhibitors. Env is a uniquely flexible molecular machine, and deep understanding of its immunogenicity and susceptibility to inhibition requires an appreciation of its atomically resolved conformational dynamics. Structural studies using truncated, solubilized, and stabilized Env constructs have yielded detailed atomic models of its main open and closed conformational states. Emerging structural studies of full-length Env support identification of asymmetric closed conformations as the “default intermediate state” (DIS), revealing details of a potentially pivotal role of quaternary asymmetry in Env conformational dynamics. At the same time, both single-molecule FRET (smFRET) and crosslinking mass spectrometry (XL-MS) of Env suggest the existence of at least one, sometimes dominant conformational state that has not been structurally characterized. This “State-1” conformation nonetheless seems relevant for both immune recognition and susceptibility to small-molecule inhibitors. We will leverage advanced Molecular Dynamics (MD) methods including targeted MD, temperature-accelerated MD, and string method to provide atomic level models for the opening of HIV-1 Env from closed (State 2) to open (State 3) conformational states and to identify critical structural changes separating State-2 from the poorly understood State-1 Env. The MD simulation methods we use will incorporate biases from multi-perspective smFRET and XL-MS, and they will in turn provide direction for expanding the set of Env constructs used in those experiments, establishing an iterative approach that progressively better defines transition mechanisms and State 1. An atomic-level understanding of HIV-1 Env conformational dynamics, identification of a yet to be structurally characterized pre-triggered conformational states, as well as the mechanism of Env activation for fusion will inform immunogen design and antiviral therapies.