Chemokine interaction with CXCR4 and ACKR3: structure and activation mechanisms

趋化因子及其受体控制着发育过程中的细胞迁移、免疫系统的动态平衡以及 对生理侮辱的炎症反应。趋化因子受体CXCR4和ACKR3也发挥关键作用 在疾病中，特别是在癌症中，它们与它们共同的趋化因子配体CXCL12作为三人组发挥作用。 CXCR4，一种典型的G蛋白偶联受体(GPCR)，通过其能力驱动肿瘤转移。 促进细胞迁移。相反，ACKR3是一种非典型受体，不与G蛋白偶联；相反，它 通过β-arrestin介导的信号，并通过偏向的β-arrestin介导的信号间接促进转移。因此， CXCR4和ACKR3是开发新型抗癌药物的有希望的靶点。 尽管它们在癌症中很重要，但这两种配体识别和激活的结构基础 CXCL12的受体仍然是一个谜。事实上，GPCR识别任何蛋白质配体的问题 直到最近，当申请者解决了CXCR4：趋化因子复合体的第一个结构时，才得到回答。 这是一项重大突破，为申请者研究分子基础做好了准备。 CXCR4和ACKR3与CXCL12的相互作用及其作为典型G蛋白偶联和β的功能 Arrestin偏向受体。申请者研究的长期目标是获得原子分辨率 了解趋化因子受体与其蛋白配体和细胞内伙伴的相互作用，从而 能够合理设计针对这些趋化因子受体的高效治疗药物。的目标是 目前的建议是理解CXCL12是如何被两个功能识别的结构基础 不同的受体以及配体结合如何转化为不同的功能反应。该提案包括 三个相辅相成的具体目标。在目标1中，CXCR4和ACKR3相互作用的结构基础 CXCL12和小分子将用X射线结晶学和实验引导的分子测定 模特儿。在目标2中，CXCR4和ACKR3激活和信号转导的药理学特性将是 通过模型指导的结合、动力学和功能实验来解卷。在《目标3》中，复杂的动态 典型的GPCRCXCR4和非典型偏向受体ACKR3的构象变化 反应配体结合的反应将使用EPR进行研究。我们全面的跨学科 方法涉及结晶学、分子建模、受体研究的广泛实验 结合和信号，以及受体构象变化和动力学的EPR表征。这个 整个项目之所以具有创新性，是因为它的结构假设、协同方法以及 这是第一次由两个膜受体组成的复杂多药理系统(其中一个是 非典型)和几个不同特异性和药理学的共享配体从结构上进行了研究 视角和在全长受体的背景下。这项研究意义重大，因为预计它将 垂直推进趋化因子受体生物学和癌症新疗法的发展。

Chemokines and their receptors control cell migration in development, in immune system homeostasis, and in inflammatory responses to physiological insults. Chemokine receptors CXCR4 and ACKR3 also play critical roles in disease, particularly cancer where they work as a trio with their mutual chemokine ligand, CXCL12. CXCR4, a canonical G-protein coupled receptor (GPCR) drives tumor metastasis by virtue of its ability to promote cell migration. In contrast, ACKR3 is an atypical receptor that does not couple to G proteins; instead it signals via β-arrestin and indirectly facilitates metastasis by biased β-arrestin mediated signaling. As such, CXCR4 and ACKR3 represent promising targets for the development of novel anti-cancer therapeutics. Despite their importance in cancer, the structural basis of ligand recognition and activation of these two receptors by CXCL12 remains a mystery. In fact, the question of recognition of any protein ligand by a GPCR was unanswered until recently when the applicants solved the first structure of a CXCR4:chemokine complex. This was a major breakthrough that poised the applicants well for studying the molecular basis of the interaction of CXCR4 and ACKR3 with CXCL12, and how they function as canonical G protein-coupled and β- arrestin biased receptors. The long term goal of the applicants' research is to obtain atomic resolution understanding of chemokine receptor interactions with their protein ligands and intracellular partners, thus enabling rational design of highly efficient therapeutics targeting these chemokine receptors. The objective of the present proposal is to understand the structural basis of how CXCL12 is recognized by two functionally distinct receptors and how ligand binding is translated into distinct functional responses. The proposal consists of three complementary Specific Aims. In Aim 1, the structural basis of CXCR4 and ACKR3 interaction with CXCL12 and small molecules will be determined using X-ray crystallography and experiment-guided molecular modeling. In Aim 2, the pharmacological peculiarities of CXCR4 and ACKR3 activation and signaling will be deconvoluted via model-guided binding, kinetic, and functional experiments. In Aim 3, the complex dynamics and the conformational changes that the canonical GPCR CXCR4 and the atypical biased receptor ACKR3 undergo in response to ligand binding will be investigated using EPR. Our comprehensive interdisciplinary approach involves crystallography, molecular modeling, a wide range of experiments for studies of receptor binding and signaling, and EPR characterization of receptor conformational changes and dynamics. The overall project is innovative because of its structural hypotheses, its synergistic methodologies, and because this is the first time a complex polypharmacological system of two membrane receptors (one of which is atypical) and several shared ligands of varying specificity and pharmacology is studied from a structural perspective and in the context of full-length receptors. The research is significant, because it is expected to vertically advance both chemokine receptor biology and the development of novel cancer therapeutics.