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gp 120的高度构象变异性。不同的实验观察表明，HIV-1包膜蛋白在与其宿主细胞受体结合时经历大的构象结构。这些构象变化导致细胞受体的Env结合位点的逐渐成熟，并且是病毒-细胞融合、细胞进入和随后的感染所需的。此外，已经鉴定了几种类型的Env抑制剂先导物，其似乎通过结合HIV-1 gp 120的未配体状态并在构象上将Env蛋白包埋到抑制受体结合位点活性的状态中而起作用。这一机构的观察认为，知识的差异的位置和动力学的未配体的gp 120诱导受体与抑制剂的构象转变，可以帮助确定抑制剂，最有效地挫败前者，而鼓励后者。然而，未配体状态本身，这在本质上是病毒包膜拮抗剂的主要结合靶点，是知之甚少，因为是从这个结构开始发生的构象变化的动力学。这些缺点将在当前的R21项目中通过使用创新策略来跟踪导致Env gp 120从unliganded到liganded形式的位点特异性构象变化来解决。R21项目有两个具体目标。在目标1中，我们将产生荧光团标记的gp 120变体，其中Env蛋白在不同的结构位置被标记，预测在与已知的包膜配体结合后改变构象，包括CD 4以及CD 4模拟和变构抑制剂。在目标2中，我们将测量配体结合至单位点标记的gp 120变体后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自明确标记的网站将被用来定义gp 120在unliganded，激活和抑制状态的构象签名。R21项目最直接的成果将是（1）衍生功能完整的gp 120变体与位置特异性荧光团报告标签和（2）证明配体诱导的构象变化可以通过荧光跟踪检测在gp 120中的空间定义的网站。从R21开始的长期研究将绘制受体活化与拮抗剂（竞争性和变构）抑制后发生的gp 120构象变化的特定位点，并获得基于荧光的抑制剂筛选，这些抑制剂诱导特异性局部构象变化，从而改善对HIV-1 Env蛋白的抑制。 公共卫生相关性：该项目将建立一种创新的多学科方法，结合蛋白质工程和荧光光谱，以扩大对HIV-1病毒外壳蛋白结构转变的理解，并长期指导识别进入抑制剂作为抗病毒剂。

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.