The structural plasticity of chemokine and gp120 recognition by CCR5

 描述（由申请人提供）：趋化因子受体CCR 5在HIV感染和疾病传播中的作用已得到充分证实;然而，CCR 5介导的HIV进入宿主细胞和抑制趋化因子进入的结构机制仍不清楚。这种知识的缺乏代表了对抗HIV的努力的关键障碍，阻碍了靶向趋化因子系统的治疗剂的合理设计并具有所需的HIV抑制谱。申请人研究的长期目标是获得CCR 5与其天然配体、药物和HIV gp 120变体相互作用的深入结构理解，从而能够合理设计高效的HIV进入抑制剂，降低对耐药性发展的敏感性。该提案的目的是阐明CCR 5与趋化因子在HIV进入抑制的背景下相互作用的结构决定因素，CCR 5与融合gp 120变体的相互作用促进HIV进入，以及CCR 5对HIV抗性和细胞向性背景下gp 120序列多样性的耐受性。核心假设是CCR 5的显著结构可塑性允许通过一组保守的结合决定簇识别不同的配体和容纳耐药HIV株。该假设是基于广泛的文献综述和在申请人的实验室中获得的数据来制定的，包括最近作为其合作的一部分解决的受体：趋化因子复合物（CXCR 4：vMIP-II）的第一个X射线结构。中心假设将通过追求两个特定目标进行检验：（1）阐明强效CCR 5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，以及（2）确定gp 120与CCR 5相互作用的结构基础以及抗性和向性机制。具体地，在目的1中，CCR 5的结构将在与趋化因子RANTES的变体的复合物中得到解决，所述趋化因子RANTES的变体通过两种不同的机制有效地抑制病毒进入：空间阻断和细胞内CCR 5的内化/隔离。这样的结构将促进对CCR 5构象的理解，所述CCR 5构象转化为特定的功能反应，并且使得能够用小分子合理地靶向这些构象。在目标2中，将应用协同计算/实验方法来获得具有合理设计的gp 120嵌合体和全长gp 120的CCR 5复合物。这一目标将有助于更好地从分子上理解艾滋病毒如何感染细胞并实现耐药性和细胞向性。整个项目是创新的，因为趋化因子和gp 120与CCR 5相互作用的结构基础尚未在全长CCR 5的背景下进行研究，并且因为将计算建模与实验紧密结合以产生这些具有挑战性的膜蛋白质的稳定，可结晶复合物的新策略：蛋白质靶点。这项研究意义重大，因为它有望垂直推进对趋化因子/受体/HIV系统的结构和机制理解，并使其组分能够合理靶向。

 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.