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三聚体（gp 160）3裂解为（gp 120/gp 41）3]。Gp 120与初级受体CD 4和辅助受体（例如， 趋化因子受体CCR 5或CXCR 4）触发gp 41中的大的结构重排， 聚变过程认为gp 120与辅助受体的相遇是gp 41重折叠的关键触发因素 事件，促进膜融合。自CCR 5和CXCR 4首次出现以来， 被鉴定为HIV-1进入的共同受体，但我们仍然没有一个清晰的画面，特别是在原子水平上， 除了一些推测性的分子模型外，我们还研究了辅助受体如何识别HIV-1 Env。的 辅助受体是具有七个跨膜片段（7 TM）的趋化因子受体，并且属于 G蛋白偶联受体（GPCR）家族。已经报道了大量修饰的晶体结构。 CCR 5和CXCR 4，揭示了这些受体的一般结构，以及它们与受体的相互作用。 各种配体，但结构未能解释这些趋化因子如何在分子上的细节， 受体作为HIV-1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 CCR 5和CXCR 4的辅助受体功能，并提供它们如何与HIV相互作用的高分辨率图片， 1 Env促进病毒进入。我们假设gp 120和 涉及多个结构元件的辅助受体是它们的高亲和力相互作用所必需的。我们有 已经纯化了HIV-1 gp 120、4结构域CD 4和未修饰的人CCR 5的稳定复合物，和 证明了进行电子显微镜（EM）研究的可行性。低温电子学的新进展 冷冻电镜（cryoEM）已经彻底改变了结构生物学领域，并产生了许多高分辨率的 结构.为了利用这些进步，我们将解决一个对世界各国都很重要的挑战性问题。 HIV和GPCR字段。我们将追求以下具体目标：1）我们将确定高分辨率结构 CD 4-gp 120-CCR 5复合物的cryoEM; 2）我们将确定高分辨率的结构， 3）我们将阐明CCR 5或CXCR 4的关键结构元件在CD 4-gp 120-CXCR 4复合物中的作用， 辅助受体功能的结构导向突变。

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.