Single-particle Reconstruction of HIV-1 Envelope Glycoprotein Trimers

描述(由申请人提供)：生物分子的详细结构的可用性可以加速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、多形性和/或构象灵活性使得它们难以用传统的方法来获得结构，如X射线结晶学或核磁共振波谱。一个这样的例子是三聚体人类免疫缺陷病毒(HIV-1)包膜(Env)糖蛋白复合体，它介导病毒进入宿主细胞。未连接的HIV-1包膜糖蛋白复合体以高能状态存在；当与CD4和CCR5受体结合时，HIV-1包膜糖蛋白呈现较低的能量构象。HIV-1包膜糖蛋白复合体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糖蛋白三聚体是进入抑制剂的主要靶点，包括小分子、多肽和中和抗体。不幸的是，构象灵活性、非连接状态的不稳定性和高度的糖基化减缓了对包膜糖蛋白三聚体的结构研究。这一知识差距是在抑制HIV-1进入和疫苗免疫原设计方面取得进展的主要障碍。拟议的研究将利用单粒子冷冻电子显微镜在近原子(3.5-5E)分辨率下获得不同构象的膜锚定HIV-1环境三聚体的结构。本应用的具体目的是：1)制备适合于单粒子冷冻电子显微镜高分辨结构测定的膜锚定的HIV-1包膜糖蛋白三聚体，并解决未连接的HIV-1包膜糖蛋白三聚体的结构；2)研究HIV-1 gp41细胞质尾部的结构及其对包膜外区结构的潜在贡献；以及3)解决HIV-1包膜糖蛋白三聚体的CD4结合构象的结构。这些研究将提供HIV-1进入过程中两个关键阶段的详细快照，为理解环境糖蛋白“膜融合机”的动态方面提供一个框架。HIV-1包膜糖蛋白复合体上保守的、功能上重要的结构将被揭示，这些结构可以作为药物或疫苗诱导的抗体的靶标。这些信息将改变我们对艾滋病毒-1进入的理解，并应激励新的干预方法。

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.