Single-particle Reconstruction of HIV-1 Envelope Glycoprotein Trimers

HIV-1 包膜糖蛋白三聚体的单粒子重建

• 批准号: 8260824
• 负责人: JOSEPH G SODROSKI
• 金额: $ 43.75万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8260824
• 关键词: Antibodies; Antibody Formation; Antigens; Area; Binding; CD4 Antigens; Cell membrane; Cells; Chemokine (C-C Motif) Receptor 5; Complex; Cryoelectron Microscopy; Crystallography; Cytoplasmic Tail; Drug Delivery Systems; Electron Microscopy; Genomics; Glycoproteins; HIV; HIV Envelope Protein gp120; HIV-1; Image; Infection; Intervention; Knowledge; Mediating; Membrane; Membrane Glycoproteins; Methods; Molecular Conformation; NMR Spectroscopy; Peptides; Potential Energy; Process; Proteins; Proteomics; Refractory; Resolution; SIV; Staging; Structure; Therapeutic; Translations; Vaccination; Vaccines; Viral; Virion; Virus; Work; conformational conversion; design; flexibility; glycosylation; inhibitor/antagonist; neutralizing antibody; novel strategies; particle; prophylactic; public health relevance; receptor binding; reconstruction; small molecule; tomography; virus envelope

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.

描述（由申请人提供）：生物分子的详细结构的可用性可以加快将基因组和蛋白质组学信息转化为治疗和预防性干预措施。不幸的是，某些蛋白质的大小，多晶和/或构象的柔韧性使它们难以合理地获得传统的获得结构的方法，例如X射线晶体学或NMR光谱。一个这样的例子是三聚体免疫缺陷病毒（HIV-1）包膜（ENV）糖蛋白复合物，它介导该病毒进入宿主细胞。非配体的HIV-1 ENV糖蛋白复合物存在于高能状态。与CD4和CCR5受体结合后，HIV-1 ENV糖蛋白假设具有较低的能量构象。 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，构象的柔韧性，非配体状态的不稳定和高度的糖基化速度减慢了对ENV糖蛋白三聚体的结构研究。知识差距代表了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光学显微镜在近原子（3.5-5 e）分辨率下以不同构象的不同构象产生膜锚定的HIV-1 env三聚体的结构。该应用的具体目的是：1）准备膜锚定的HIV-1 env糖蛋白三聚体，这些三聚体适合通过单个粒子冷冻微观显微镜测定高分辨率结构，并解决无配合的HIV-1 envimer的结构； 2）研究HIV-1 GP41细胞质尾巴的结构及其对Env ectodomain结构的潜在贡献； 3）解决HIV-1 Env糖蛋白夹层的CD4结合构象的结构。这些研究将在HIV-1输入过程中产生两个关键阶段的详细快照，从而提供了一个框架，以理解ENV糖蛋白“膜融合机”的动态方面。将揭示HIV-1 Env糖蛋白复合物上可以作为药物或疫苗诱导的抗体靶标的保守，功能上重要的结构。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）包膜糖蛋白尖峰介导该病毒进入宿主细胞，因此是抑制剂和疫苗的有吸引力的靶标。但是，缺乏有关HIV-1包膜糖蛋白峰值结构的信息，这是该领域进步的主要障碍。我们将开发电子显微镜中的方法，以实现HIV-1包膜糖蛋白尖峰的详细结构蓝图。