Viral GPCR recognition of chemokines and engineered ligands

摘要： 尽管在了解G蛋白偶联的结构-功能特性方面取得了很大的进展 作为小分子受体的受体(Gpr)仍然缺乏知识。 关于与蛋白质配体结合的GPCRs。有几类gpr对蛋白质有反应。 目前还不清楚识别和受体激活的机制与这些机制的关系 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 几乎只针对小分子gpcr，然而许多治疗机会存在于 通过GPCR发出信号。使用gpr作为信号受体的最大一类蛋白质是 趋化因子，与趋化因子大家族gpr结合并通过其传递信号。趋化因子是 强大的免疫调节剂，在炎症、感染期间以及它们的活动中发挥重要作用 与许多人类疾病有关。一些病毒被劫持并被“改变用途” 趋化因子gpr用于免疫逃避和增强毒力。病毒gpr可介导致癌作用 这一转变导致了一系列临床问题。P.I.S实验室最近做出了一项 通过确定一个化合物的晶体结构，我们对趋化因子gpr结构的了解取得了进展。 病毒编码的(人巨细胞病毒-巨细胞病毒)GPCR，US28，与人趋化因子结合 CX3CL1(Fractalkine)。US28拥有许多有趣的功能特性，使其成为一种优秀的 作为一个整体，探索趋化因子GPCRs的机制和结构-功能特性的系统。为 例如，US28是结构性活性的，这对HCMV的致病很重要，也是唯一的 趋化因子GPCR可同时作用于CXC和CC类趋化因子。US28也用作条目 人类免疫缺陷病毒1型(HIV-1)联合受体gp120，通常使用人类 趋化因子GPCRCXCR4和CCR5作为进入受体。US28具有特别有利的生化 与其他趋化因子受体相比，它的特性使其成为一个出色的结构系统- 功能分析。我们有强大的生化和结构通道进入US28，已经开发出高 水平表达协议，分离羊驼纳米体，并有能力结晶不同的 US28与趋化因子和gp120的复合体。在这里，我们建议在US28上合并结构性努力- 趋化因子，US28-gp120和US28 G蛋白复合体，结合组合生物学设计新的 趋化因子和蛋白质为基础的替代配体的美国28。这些研究将共同探索 结构基础US28-趋化因子识别和信号特性，阐明gp120是如何感染HIV的 利用趋化因子gpr作为辅助受体，探索工程化治疗的新机会 具有偏向信号特性的趋化因子gpr配体。

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.