The structural plasticity of chemokine and gp120 recognition by CCR5

 描述(由申请人提供)：趋化因子受体CCR5在艾滋病毒感染和疾病传播中的作用是众所周知的；然而，CCR5介导的艾滋病毒进入宿主细胞和抑制趋化因子进入的结构机制仍然不清楚。这种知识的缺乏是抗击艾滋病毒努力的关键障碍，阻碍了针对趋化因子系统的治疗方法的合理设计，并具有所需的艾滋病毒抑制特征。申请者研究的长期目标是深入了解CCR5与其天然配体、药物和HIV gp120变体的相互作用，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。本研究的目的是阐明CCR5与趋化因子在抑制HIV进入的背景下相互作用的结构决定因素，CCR5与促进HIV进入的融合基因gp120变体的相互作用，以及在HIV抗性和细胞趋向性的背景下CCR5对gp120序列多样性的耐受性。中心假设是，CCR5显着的结构可塑性允许识别不同的配体，并通过一组保守的结合决定子适应耐药的艾滋病毒株。这一假设是基于广泛的文献综述和申请者实验室获得的数据，包括最近作为他们合作的一部分解决的受体：趋化因子复合体(CXCR4：vMIP-II)的第一个X射线结构。中心假设将通过追求两个特定的目标来检验：(1)阐明有效的CCR5结合趋化因子的亲和力、特异性和抗病毒活性的结构决定因素；(2)确定gp120与CCR5相互作用的结构基础以及耐药和趋化机制。具体地说，在目标1中，CCR5的结构(S)将与趋化因子RANTES的变体形成复合体，通过两种不同的机制有效地抑制病毒进入：空间阻断和CCR5在细胞内的内化/隔离。这种结构将促进对CCR5构象的理解，这些构象转化为特定的功能反应，并使这些构象能够合理地与小分子打靶。在目标2中，将采用协同计算/实验方法来获得具有合理设计的gp120嵌合体和全长gp120的CCR5络合物。这一目标将有助于更好地从分子上了解艾滋病毒是如何感染细胞并实现耐药性和细胞趋向性的。整个项目是创新的，因为趋化因子和gp120与CCR5相互作用的结构基础还没有在全长CCR5的背景下进行研究，而且因为新的策略将计算建模与实验高度结合起来，以产生这些具有挑战性的膜-蛋白质：蛋白质靶标的稳定、可结晶的复合体。这项拟议的研究具有重要意义，因为它有望从结构和机制上促进对趋化因子/受体/艾滋病毒系统的理解，并使其能够合理靶向其组成部分。

 DESCRIPTION (provided by applicant): The role of the chemokine receptor CCR5 in HIV infection and disease transmission is well- established; however, the structural mechanisms of CCR5-mediated HIV entry into host cells and inhibition of entry by chemokines remain elusive. This lack of knowledge represents a critical barrier for efforts to combat HIV, hindering rational design of therapeutics targeting the chemokine system and possessing desired HIV inhibition profiles. The long term goal of the applicants' research is to obtain a deep structural understanding of CCR5 interactions with its natural ligands, drugs, and HIV gp120 variants, thus enabling rational design of highly efficient HIV entry inhibitors with reduced susceptibility to development of resistance. The objective of this proposal is to elucidate the structural determinants of the interaction of CCR5 with chemokines in the context of HIV entry inhibition, the interaction of CCR5 with fusogenic gp120 variants facilitating HIV entry, and the tolerance of CCR5 to gp120 sequence diversity in the context of HIV resistance and cellular tropism. The central hypothesis is that the remarkable structural plasticity of CCR5 allows for recognition of diverse ligands and accommodation of drug-resistant HIV strains through a conserved set of binding determinants. This hypothesis has been formulated based on extensive literature review and data obtained in the applicants' laboratories including the first X-ray structure of a receptor:chemokine complex (CXCR4:vMIP-II) that was recently solved as a part of their collaboration. The central hypothesis will be tested by pursuing two Specific Aims: (1) Elucidate the structural determinants of affinity, specificity and antiviral activity of potent CCR5 binding chemokines, and (2) Determine the structural basis of the interaction of gp120 with CCR5 and the mechanisms of resistance and tropism. Specifically, in Aim 1, structure(s) of CCR5 will be solved in complex with variants of the chemokine RANTES that potently inhibit viral entry by two different mechanisms: steric blockade and internalization/sequestration of CCR5 inside cells. Such structures will promote understanding of CCR5 conformations that translate into specific functional responses and enable rational targeting of these conformations with small molecules. In Aim 2, a synergistic computational/experimental approach will be applied to obtain CCR5 complexes with rationally designed gp120 chimeras and full-length gp120. This aim will contribute to a better molecular understanding of how HIV infects cells and achieves resistance and cellular tropism. The overall project is innovative because the structural basis of chemokine and gp120 interactions with CCR5 has not been studied in the context of full length CCR5, and because of novel strategies that heavily integrate computational modeling with experiments to generate stable, crystallizable complexes of these challenging membrane-protein:protein targets. The proposed research is significant because it is expected to vertically advance structural and mechanistic understanding of the chemokine/receptor/HIV system and enable rational targeting of its components.