Structural Basis of Coreceptor Recognition by HIV-1 Envelope Spike

项目摘要 HIV感染开始于病毒和靶细胞膜的融合，由病毒包膜糖蛋白驱动 [env；三聚(Gp160)3裂解为(gp120/gp41)3]。Gp120与主要受体CD4和辅受体结合(例如： 趋化因子受体CCR5或CXCR4)触发gp41的大规模结构重排，从而驱动细胞膜 核聚变过程。Gp120与辅助受体的相遇被认为是gp41折叠的关键触发因素。 事件，这促进了膜融合。从CCR5和CXCR4第一次出现到现在，已经过去了二十年 被确认为HIV-1进入的辅助受体，但我们仍然没有一个清晰的图景，特别是在原子 除了一些推测的分子模型外，共受体如何识别HIV-1环境的分辨率。这个 辅受体是具有七个跨膜片段(7TM)的趋化因子受体，属于 G蛋白偶联受体(GPCRs)家族。据报道，晶体结构经过了大量的修饰 CCR5和CXCR4，揭示了这些受体的一般结构以及它们与 不同的配体，但结构不能解释这些趋化因子是如何 受体作为HIV-1的辅助受体发挥作用。在这项提案中，我们计划更好地了解艾滋病毒-1 CCR5和CXCR4的共同受体功能，并提供它们如何与艾滋病毒相互作用的高分辨率图片- 1环境促进病毒进入。我们假设gp120和gp120之间的广泛接口 涉及多个结构元素的辅受体是它们高亲和力相互作用所必需的。我们有 已经提纯了稳定的HIV-1gp120，4结构域CD4和未经修饰的人CCR5复合体，以及 论证了开展电子显微镜(EM)研究的可行性。低温电子的最新进展 显微镜(低温电子显微镜)给结构生物学领域带来了革命性的变化，产生了无数高分辨率的 结构。为了利用这些进展，我们将解决一个具有挑战性的问题，这对双方都很重要 HIV和GPCR域。我们将追求以下具体目标：1)确定高分辨率结构 2)我们将确定CD_4-gp120-CCR5的高分辨结构 3)我们将阐明CCR5或CXCR4的关键结构元件在 辅受体通过结构导向突变发挥作用。

Project Summary HIV infection begins with fusion of viral and target cell membranes, driven by the viral envelope glycoprotein [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3]. Gp120 binding to primary receptor CD4 and coreceptor (e.g. chemokine receptor CCR5 or CXCR4) trigger large structural rearrangements in gp41 that drive the membrane fusion process. Encounter of the coreceptor by gp120 is believed to be the crucial trigger for gp41 refolding events, which promotes membrane fusion. It has been two decades since CCR5 and CXCR4 were first identified as the coreceptors for HIV-1 entry, but we still do not have a clear picture, in particular, at atomic resolution, of how the coreceptor recognizes HIV-1 Env, except for some speculative molecular modeling. The coreceptors are chemokine receptors with seven transmembrane-spanning segments (7TMs) and belong to the family of G protein-coupled receptors (GPCRs). Crystal structures have been reported for heavily modified CCR5 and CXCR4, revealing the general architecture of these receptors, as well as their interactions with the various ligands, but the structures fall short of explaining the molecular details of how these chemokine receptors function as HIV-1 coreceptors. In this proposal, we plan to gain a better understanding of the HIV-1 coreceptor function of CCR5 and CXCR4 and to provide high-resolution pictures of how they interact with HIV- 1 Env to promote viral entry. We hypothesize that an extensive interface between gp120 and the coreceptor involving multiple structural elements is required for their high-affinity interaction. We have already purified a stable complex of HIV-1 gp120, 4 domain CD4 and an unmodified human CCR5, and demonstrated the feasibility to carry out electron microscopy (EM) studies. Recent advances in cryo-electron microscopy (cryoEM) have revolutionized the field of structural biology and produced numerous high-resolution structures. To capitalize on these advances, we will tackle a challenging problem that is important to both the HIV and GPCR fields. We will pursue following specific aims: 1) we will determine the high-resolution structure of the complex of CD4-gp120-CCR5 by cryoEM; 2) we will determine the high-resolution structure of the complex of CD4-gp120-CXCR4; 3) we will elucidate the role of key structural elements of CCR5 or CXCR4 in coreceptor function by structure-guided mutagenesis.