Single-particle Reconstruction of HIV-1 Envelope Glycoprotein Trimers

描述（由申请人提供）：生物分子详细结构的可用性可以加快基因组和蛋白质组信息转化为治疗和预防干预措施。不幸的是，一些蛋白质的大小、多晶性和/或构象灵活性使得它们难以用常规方法获得结构，例如X射线晶体学或NMR光谱学。一个这样的实例是三聚体人免疫缺陷病毒（HIV-1）包膜（Env）糖蛋白复合物，其介导病毒进入宿主细胞。未配体的HIV-1 Env糖蛋白复合物以高能状态存在;在与CD 4和CCR 5受体结合后，HIV-1 Env糖蛋白呈现低能构象。HIV-1 Env糖蛋白复合物中的这些构象转变最终导致病毒和靶细胞膜的融合。作为暴露在HIV-1膜上的唯一病毒特异性蛋白，Env糖蛋白三聚体代表了进入抑制剂的主要靶标，包括小分子、肽和中和抗体。不幸的是，构象的灵活性，unliganded状态的不稳定性，和高度的糖基化减缓了Env糖蛋白三聚体的结构研究。这种知识上的差距是HIV-1进入抑制和疫苗免疫原设计进展的主要障碍。拟议的研究将利用单粒子冷冻电子显微镜以近原子（3.5 - 5 E）分辨率产生不同构象的膜锚定HIV-1 Env三聚体结构。本申请的具体目的是：1）制备膜锚定的HIV-1 Env糖蛋白三聚体，其适用于通过单颗粒冷冻电子显微镜进行高分辨率结构测定，并解析未配体的HIV-1 Env三聚体的结构; 2）研究HIV-1 gp 41胞质尾区的结构及其对Env胞外域结构的潜在贡献; 3）解析HIV-1 Env糖蛋白三聚体的CD 4结合构象。这些研究将产生HIV-1进入过程中两个关键阶段的详细快照，为理解Env糖蛋白“膜融合机器”的动态方面提供框架。HIV-1 Env糖蛋白复合物上保守的、功能重要的结构将被揭示，这些结构可以作为药物或疫苗诱导抗体的靶点。这些信息将改变我们对HIV-1进入的理解，并应激发新的干预方法。 公共卫生相关性：人类免疫缺陷病毒（HIV-1）包膜糖蛋白刺突介导病毒进入宿主细胞，因此是抑制剂和疫苗的有吸引力的靶标。然而，缺乏关于HIV-1包膜糖蛋白刺突结构的信息一直是该领域进展的主要障碍。我们将开发电子显微镜的方法，使详细的结构蓝图的HIV-1包膜糖蛋白刺实现。

DESCRIPTION (provided by applicant): The availability of detailed structures of biomolecules can expedite the translation of genomic and proteomic information into therapeutic and prophylactic interventions. Unfortunately, the size, pleiomorphism and/or conformational flexibility of some proteins renders them refractory to conventional approaches to obtaining structure, such as x-ray crystallography or NMR spectroscopy. One such example is the trimeric human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein complex, which mediates the entry of the virus into the host cell. The unliganded HIV-1 Env glycoprotein complex exists in a high-energy state; upon binding to the CD4 and CCR5 receptors, the HIV-1 Env glycoproteins assume lower-energy conformations. These conformational transitions in the HIV-1 Env glycoprotein complex ultimately result in the fusion of the viral and target cell membranes. As the only virus-specific protein exposed on the HIV-1 membrane, the Env glycoprotein trimer represents a major target for entry inhibitors, including small molecules, peptides and neutralizing antibodies. Unfortunately, conformational flexibility, the lability of the unliganded state, and a high degree of glycosylation have slowed structural studies of the Env glycoprotein trimer. This gap in knowledge represents a major barrier to progress in HIV-1 entry inhibition and vaccine immunogen design. The proposed studies will utilize single-particle cryoelectron microscopy to yield structures of membrane-anchored HIV-1 Env trimers in different conformations at near-atomic (3.5 - 5 E) resolution. The Specific Aims of this application are: 1) To prepare membrane-anchored HIV-1 Env glycoprotein trimers that are suitable for high-resolution structure determination by single-particle cryoelectron microscopy and to solve the structure of the unliganded HIV-1 Env trimer; 2) To investigate the structure of the HIV-1 gp41 cytoplasmic tail and its potential contribution to the structure of the Env ectodomain; and 3) To solve the structure of the CD4-bound conformation of the HIV-1 Env glycoprotein trimer. These studies will yield detailed snapshots of two key stages in the process of HIV-1 entry, providing a framework for understanding the dynamic aspects of the Env glycoprotein "membrane-fusing machine". Conserved, functionally important structures on the HIV-1 Env glycoprotein complex that can serve as targets for drugs or vaccine-induced antibodies will be revealed. This information will transform our understanding of HIV-1 entry and should inspire new approaches to intervention. PUBLIC HEALTH RELEVANCE: The human immunodeficiency virus (HIV-1) envelope glycoprotein spike mediates the entry of the virus into the host cell, and therefore is an attractive target for inhibitors and vaccines. However, lack of information about the structure of the HIV-1 envelope glycoprotein spike has been a major barrier to progress in this area. We will develop methods in electron microscopy that allow a detailed structural blueprint of the HIV-1 envelope glycoprotein spike to be realized.