HIV-1 gp120 Conformational Transitions in Activation and Antagonism

HIV-1 gp120 激活和拮抗作用中的构象转变

基本信息

批准号：
8238278
负责人：
CAMERON F ABRAMS
金额：
$ 18.95万
依托单位：
DREXEL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8238278
关键词：
Achievement Address Anisotropy Antiviral Agents Binding Binding Sites Calorimetry Capsid Proteins Cell fusion Cells Crystallization Crystallography Data Analyses Derivation procedure Fluorescence Fluorescence Anisotropy Fluorescence Spectroscopy Glycoproteins HIV Envelope Protein gp120 HIV-1 Infection Knowledge Label Lead Ligand Binding Ligands Location Maps Measurement Measures Methods Molecular Conformation Outcome Property Protein Engineering Protein Region Proteins Rationalization Reagent Receptor Activation Receptor Cell Recombinant Proteins Refractory Reporter Research Signal Transduction Site Structural Models Structure Testing Time Variant Viral Virus base conformational conversion design electron tomography env Gene Products flexibility fluorophore improved inhibitor/antagonist innovation insight interdisciplinary approach mutant novel public health relevance receptor receptor binding stem tool

项目摘要

DESCRIPTION (provided by applicant): This project addresses a major barrier in designing HIV-1 entry inhibitors that stems from the high conformational variability of the viral envelope glycoprotein gp120. Diverse experimental observations have shown that the HIV-1 envelope protein undergoes large conformational structuring upon binding to its host cell receptors. These conformational changes lead to progressive maturation of Env binding sites for the cell receptors and are required for virus-cell fusion, cell entry and consequent infection. In addition, several types of Env inhibitor leads have been identified that appear to function by binding the unliganded state of HIV-1 gp120 and conformationally entrapping Env protein into states that have suppressed receptor binding site activity for cell entry. This body of observations argues that knowledge of differences in the locations and dynamics of conformational transitions of unliganded gp120 induced by receptors vs inhibitors could help to identify inhibitors that most effectively frustrate the former while encouraging the latter. Yet, the unliganded state itself, which is in essence the primary binding target for antagonists of viral envelope, is poorly understood, as are the dynamics of conformational changes that occur starting from this structure. These shortcomings will be addressed in the current R21 project by using innovative strategies to track site-specific conformational changes leading from unliganded to liganded forms of Env gp120. The R21 project will have two specific aims. In Aim 1, we will produce fluorophore-tagged gp120 variants in which the Env protein is labeled in different structural locations predicted to change conformations upon binding to known envelope ligands, including CD4 and both CD4-mimicking and allosteric inhibitors. In Aim 2, we will measure location- specific changes in both time-resolved and steady state fluorescence anisotropy upon ligand binding to single- site labeled gp120 variants. Experimental anisotropy curves derived from distinctly labeled sites will be used to define conformational signatures of gp120 in unliganded, activated and inhibited states. The most immediate outcomes of the R21 project will be (1) derivation of functionally intact gp120 variants with location-specific fluorophore reporter tags and (2) demonstration that ligand-induced conformational changes can be detected at spatially defined sites in gp120 through fluorescence tracking. Longer term research following up from the R21 will map specific sites of conformational changes in gp120 that occur upon receptor activation vs antagonist (competitive as well as allosteric) inhibition and derive fluorescence-based screens for inhibitors that induce specifically localized conformational changes leading to improved inhibition of HIV-1 Env protein. PUBLIC HEALTH RELEVANCE: This project will establish an innovative multidisciplinary approach, combining protein engineering and fluorescence spectroscopy, to expand understanding of the structural transitions of HIV-1 virus coat protein and long term guide identification of entry inhibitors as antiviral agents.

描述（由申请人提供）：该项目解决了设计HIV-1进入抑制剂的主要障碍，该抑制剂源于病毒包膜糖蛋白GP120的高构象变异性。各种实验观察结果表明，HIV-1包膜蛋白在与其宿主细胞受体结合后经历了大构象结构。这些构象变化导致细胞受体的ENV结合位点的逐渐成熟，这是病毒细胞融合，细胞进入和随之而来的感染所必需的。此外，已经确定了几种类型的ENV抑制剂引线，它们通过结合HIV-1 GP120的无配置状态并构象将ENV蛋白纳入抑制细胞进入受体结合位点活性的状态。这一观察结果表明，对受体与抑制剂引起的非配体GP120构象转变位置和动力学的差异知识可以帮助鉴定抑制剂，这些抑制剂在鼓励后者的同时最有效地挫败了前者。然而，本质上，非配体的状态本身是病毒包膜拮抗剂的主要结合靶标，也是很众所周知的，从这种结构开始发生的构象变化的动力学也是如此。这些缺点将在当前的R21项目中解决，通过使用创新策略来跟踪特定地点的构象变化，从无配合到ENV GP120的配体形式。 R21项目将有两个具体的目标。在AIM 1中，我们将产生具有荧光团标签的GP120变体，其中ENV蛋白在与已知包络配体结合后（包括CD4和CD4模拟和变构抑制剂）结合后被预测会改变构象的不同结构位置标记。在AIM 2中，我们将在配体与单位位点标记的GP120变体结合后测量时间分辨和稳态荧光各向异性的位置特异性变化。从明显标记的位点得出的实验各向异性曲线将用于定义在无物体，激活和抑制状态下GP120的构象特征。 R21项目的最直接结果将是（1）具有特定于位置的荧光团记者标签的功能完整的GP120变体，并且（2）证明可以通过荧光跟踪在GP120中的空间定义的位点检测配体诱导的构象变化。从R21进行后续进行的长期研究将绘制GP120中构象变化的特定位点，这些位点是受体激活与拮抗剂（竞争性和变构者）抑制作用，并得出基于荧光的筛选抑制剂的抑制剂，这些抑制剂可诱导特定局部构型变化，从而改善HIV-1 Envy蛋白的抑制作用。公共卫生相关性：该项目将建立一种创新的多学科方法，将蛋白质工程和荧光光谱结合在一起，以扩大对HIV-1病毒外套蛋白质的结构过渡的理解以及将入口抑制剂作为抗病毒剂的长期指导鉴定。