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

项目摘要 HIV-1包膜糖蛋白刺突（Env）介导病毒进入靶细胞。因为环境是唯一 作为病毒体表面的病毒蛋白，它对潜在疫苗和小分子疫苗的开发至关重要。 进入抑制剂Env是一种独特的柔性分子机器，对其免疫原性的深入了解 而对抑制的敏感性需要对其原子分辨的构象动力学的理解。 使用截短的、溶解的和稳定的Env构建体的结构研究已经产生了详细的原子结构。 其主要的开放和封闭构象状态的模型。全长Env的新兴结构研究 支持非对称闭合构象作为“默认中间态”（DIS）的鉴定，揭示了 详细的潜在的关键作用，四元不对称性的Env构象动力学。与此同时， Env的单分子FRET（smFRET）和交联质谱（XL-MS）都表明 存在至少一个，有时占主导地位的构象状态，尚未在结构上 表征了尽管如此，这种“状态-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 包括目标MD、温度加速MD和弦方法，以提供 将HIV-1 Env从封闭（状态2）构象状态打开为开放（状态3）构象状态，并鉴定关键的 结构变化将State-2与知之甚少的State-1 Env分开。MD模拟方法， 使用将结合来自多角度smFRET和XL-MS的偏差，它们将反过来提供方向 为了扩展这些实验中使用的Env构建体的集合，建立了一种迭代方法， 逐步更好地定义过渡机制和状态1。对HIV-1 Env的原子级理解 构象动力学，识别尚未结构表征的预触发构象 状态，以及Env激活融合的机制将为免疫原设计和抗病毒治疗提供信息。 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.