Dissecting the interactions of gp120 with chemokine receptor CXCR4

 描述(申请人提供)：艾滋病毒目前影响全球3500万人。感染是通过HIV颗粒表面的包膜糖蛋白gp120与CD4以及宿主细胞上两个趋化因子受体之一CXCR4或CCR5的相互作用而发生的，CXCR4或CCR5通过gp41介导的病毒和宿主细胞膜的融合促进病毒进入。嗜CCR5病毒主要负责疾病的初始感染和传播，而嗜CXCR4病毒与艾滋病的后期进展有关。因此，自1981年以来，已经做出了重大努力来开发有效的艾滋病毒辅助受体的小分子、多肽和蛋白质“进入抑制剂”，并于2007年批准了CCR5进入抑制剂马拉韦罗。CXCR4嗜性病毒的出现在约50%的感染者中，它与CD4+细胞计数更快下降和更快进展到艾滋病症状阶段的相关性也促进了CXCR4抑制剂的开发。关于gp120如何与CXCR4相互作用进入细胞的结构信息对于推动这一努力以及提供对病毒进入、趋向性和耐药性的更好理解将是非常有价值的。虽然gp120的结构已经与参与HIV进入细胞的其他可溶性蛋白一起解决了，但对其与完整 目前尚缺乏长度的趋化因子辅助受体。这是由于确定涉及膜蛋白的结构的挑战，特别是与趋化因子和gp120等蛋白质配体有关的结构。最近，我们解决了第一个CXCR4与蛋白质配体-病毒趋化因子vMIP-II的复合体的结构。带有HIV蛋白配体gp120的CXCR4的结构代表了这些研究的明显扩展。我们的长期目标是从结构上了解CCR5和CXCR4与HIV gp120的相互作用，从而能够合理设计高效的HIV进入抑制剂，降低对耐药性的易感性。因此，本提案的目标是产生全长CXCR4与gp120的稳定络合物，作为未来结构研究的前奏。我们的中心假设是，gp120与辅助受体的相互作用在结构上模仿了趋化因子的相互作用，并且至少由两个不同的表位介导。我们将专注于以决定gp120 V3环的取向为中心的结合决定因素。此外，我们将使用在确定CXCR4：vMIP-II结构的过程中开发的计算指导的“二硫键陷阱”策略来识别引入gp120和CXCR4的非天然半胱氨酸之间自发形成的二硫键，以便使稳定的CXCR4：gp120络合物适合结构研究。在此过程中，我们将确定可用于对相互作用的复合体进行建模的距离限制，并开发一个平台，以了解CXCR4在HIV抗性和趋向性的背景下对gp120序列多样性的耐受性。这项提议在其具有挑战性的目标和新的二硫化物陷阱方法方面都是创新的，它具有重要意义，因为它将促进对艾滋病毒进入、趋向性、耐药性和治疗设计的理解。

 DESCRIPTION (provided by applicant): HIV currently affects 35 million people worldwide. Infection occurs by the interaction of the envelope glycoprotein gp120 on the surface of the HIV particle with CD4 and one of two chemokine receptors, CXCR4 or CCR5, on host cells, which promotes viral entry by gp41-mediated fusion of viral and host cell membranes. CCR5-tropic viruses are primarily responsible for initial infection and transmission of the disease, while CXCR4-tropic viruses are correlated with later stage progression to full-blown AIDS. Consequently, since 1981, significant efforts have been deployed to develop potent small molecule, peptide and protein "entry inhibitors" of HIV coreceptors, and a CCR5 entry inhibitor, Maraviroc, was approved in 2007. The emergence of CXCR4 tropic virus in ~50% percent of infected individuals and its correlation with more rapid decline in CD4+ cell counts and faster progression to the symptomatic stage of AIDS has also encouraged development of CXCR4 inhibitors. Structural information on how gp120 interacts with CXCR4 to enter cells would be highly valued to advance such endeavors, as well as to provide a better understanding of viral entry, tropism and resistance. While structures of gp120 have been solved with other soluble proteins involved in entry of HIV into cells, structural understanding of its interaction with full length chemokine coreceptors is still lacking. This is due to the challenge of determining structures involving membrane proteins, particularly with protein ligands like chemokines and gp120. Recently, we solved the structure of the first complex of CXCR4 with a protein ligand, the viral chemokine vMIP-II. Structures of CXCR4 with the HIV protein ligand, gp120, represent an obvious extension of these studies. Our long term goal is to obtain a structural understanding of CCR5 and CXCR4 interactions with HIV gp120, thus enabling rational design of highly efficient HIV entry inhibitors with reduced susceptibility to development of resistance. The objective of the present proposal is therefore to generate stable complexes of full length CXCR4 with gp120 as a prelude to future structural studies. Our central hypothesis is that gp120 interaction with the co-receptors structurally mimics that of chemokines and is mediated by at least two distinct epitopes. We will focus on binding determinants centered on the tropism determining gp120 V3 loop. Furthermore, we will use a computationally-guided "disulfide trap" strategy developed in the course of determining the CXCR4:vMIP-II structure to identify disulfides that spontaneously form between non-native cysteines introduced into gp120 and CXCR4 in order to make stable CXCR4:gp120 complexes suitable for structural studies. In the process of doing so, we will determine distance restraints that can be used to model complexes of the interaction, and develop a platform for understanding the tolerance of CXCR4 to gp120 sequence diversity in the context of HIV resistance and tropism. The proposal is innovative in both its challenging objectives and its novel disulfide trap methodology, and it is significant because it will advance the understanding of HIV entry, tropism, resistance and therapeutic design.