Viral GPCR recognition of chemokines and engineered ligands

摘要： 尽管在理解G蛋白偶联的结构-功能特性方面取得了很大进展， 受体（GPCR）作为小分子的受体，仍然缺乏知识 关于GPCR与蛋白质配体的关系。有几类GPCR对蛋白质产生反应， 目前还不清楚识别和受体激活的机制如何与配体的那些相关。 小分子配体。这是一个需要澄清的重要问题，因为药物发现工作的重点是 几乎完全依赖于小分子GPCR，但对于 通过GPCR发出信号。使用GPCR作为信号受体的最大一类蛋白质是 趋化因子，其结合至趋化因子GPCR的大家族并通过其发出信号。趋化因子是 强效免疫调节剂，在炎症、感染期间及其活动中发挥重要作用 与许多人类疾病有关。一些病毒已经劫持和“重新利用” 趋化因子GPCR用于免疫逃避和增强毒力。病毒GPCR可介导致癌 转化并导致一系列临床问题。私家侦探的实验室最近做了一个 通过确定一种趋化因子GPCR的晶体结构， 病毒编码的（人巨细胞病毒- HCMV）GPCR，US 28，与人趋化因子结合 CX3CL1（Fractalkine）。US 28具有许多有趣的功能特性，使其成为一种优异的 系统来探测趋化因子GPCR作为一个整体的机制和结构-功能特性。为 例如，US 28是组成型活性的，这对于HCMV发病机制是重要的，并且是唯一的 趋化因子GPCR结合CXC和CC类趋化因子。US 28也是一个入口 人免疫缺陷病毒1型（HIV-1）gp 120的共受体，其通常使用人 趋化因子GPCR、CXCR 4和CCR 5作为进入受体。US 28具有特别有利的生物化学特性， 与其他趋化因子受体相比，其特性使其成为结构- 功能分析我们有强大的生化和结构访问US 28，已经开发了一个高 水平表达方案，分离的Alpaca纳米抗体，并具有结晶不同的能力， US 28与趋化因子和gp 120的复合物。在这里，我们建议合并US 28的结构性工作- 趋化因子，US 28-gp 120和US 28 G-蛋白复合物，与组合生物学工程新的 趋化因子和基于蛋白质的US 28替代配体。这些研究将共同探讨 结构基础US 28-趋化因子识别和信号传导特性，阐明gp 120 HIV 将趋化因子GPCR作为共受体，并探索新的工程治疗机会 趋化因子GPCR配体具有偏置信号传导特性。

Abstract: Despite a great deal of progress in understanding the structure-function properties of G protein-coupled receptors (GPCR) that serve as receptors for small molecules, there remains a dearth of knowledge about GPCRs that engage protein ligands. There are several classes of GPCR that respond to protein ligands and it is unclear how the mechanisms of recognition and receptor activation relate to those of small molecule ligands. This is an important issue to clarify given that drug discovery efforts are focused almost exclusively on small molecule GPCR, yet many therapeutic opportunities exist for proteins that signal through GPCR. The largest class of proteins that use GPCR as signaling receptors are chemokines, which bind to and signal through a large family of chemokine GPCR. Chemokines are potent immune-modulators, play important roles in inflammation, during infection, and their activities have been implicated in many human diseases. Some viruses have hijacked and `repurposed' chemokine GPCR for immune evasion and to enhance virulence. Viral GPCR can mediate oncogenic transformation and contribute to an array of clinical problems. The P.I.'s laboratory recently made an advance in our understanding of chemokine GPCR structure, by determining the crystal structure of a virally encoded (Human cytomegalovirus - HCMV) GPCR, US28, bound to the human chemokine CX3CL1 (Fractalkine). US28 possesses many interesting functional properties that make it an excellent system to probe mechanisms and structure-function properties of chemokine GPCRs as a whole. For example, US28 is constitutively active, which is important for HCMV pathogenesis, and is the only chemokine GPCR to engage both CXC and CC classes of chemokines. US28 also serves as an entry co-receptor for human immunodeficiency virus type 1 (HIV-1) gp120, which typically uses the human chemokine GPCR CXCR4 and CCR5 as entry receptors. US28 has particularly favorable biochemical properties compared to other chemokine receptors that make it an outstanding system for structure- function analysis. We have robust biochemical and structural access to US28, having developed a high level expression protocol, isolated Alpaca nanobodies, and have the ability to crystallize different complexes of US28 with chemokines and gp120. Here we propose to merge structural efforts on US28- chemokine, US28-gp120, and US28 G-protein complexes, with combinatorial biology to engineer novel chemokine and protein-based surrogate ligands for US28. These studies will collectively probe the structural basis US28-chemokine recognition and signaling properties, elucidate how gp120 HIV engages chemokine GPCR as co-receptors, and explore new therapeutic opportunities for engineering chemokine GPCR ligands with biased signaling properties.