HIV-1 gp120 Conformational Transitions in Activation and Antagonism

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，该障碍源于病毒包膜糖蛋白gp120的高构象变异性。不同的实验观察表明，HIV-1包膜蛋白在与宿主细胞受体结合时经历了大的构象结构。这些构象变化导致细胞受体的Env结合位点逐渐成熟，并且是病毒与细胞融合、细胞进入和随后的感染所必需的。此外，已经确定了几种类型的Env抑制剂导联，它们似乎通过结合HIV-1 gp120的非配体状态和构象包绕Env蛋白进入抑制受体结合位点活性的状态来起作用。这些观察认为，了解受体与抑制剂诱导的无配体gp120构象转变的位置和动力学差异，有助于确定最有效地抑制前者而鼓励后者的抑制剂。然而，非配体状态本身，本质上是病毒包膜拮抗剂的主要结合靶标，人们对其了解甚少，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项目中得到解决，通过使用创新的策略来跟踪Env gp120从非配体到配体形式的位点特异性构象变化。R21项目将有两个具体目标。在Aim 1中，我们将生产荧光团标记的gp120变体，其中Env蛋白被标记在不同的结构位置，预测在与已知的包膜配体结合时改变构象，包括CD4和CD4模拟和变构抑制剂。在目标2中，我们将测量配体结合单位点标记gp120变体时时间分辨和稳态荧光各向异性的位置特异性变化。实验各向异性曲线来源于明显标记的位点，将用于定义gp120在未配体、激活和抑制状态下的构象特征。R21项目最直接的成果将是：(1)衍生出具有位置特异性荧光团报告标签的功能完整的gp120变体；(2)证明通过荧光跟踪可以在gp120的空间定义位点检测到配体诱导的构象变化。R21后续的长期研究将绘制gp120在受体激活与拮抗剂（竞争性和变构性）抑制时发生的构象变化的特定位点，并获得基于荧光的抑制剂筛选，这些抑制剂可诱导特异性定位构象变化，从而改善HIV-1 Env蛋白的抑制。

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.