Hydrogen Deuterium Exchange Mapping of Chemokine Receptor Complex Interfaces

描述（由申请方提供）：趋化因子是免疫监视、淋巴细胞发育和炎症过程中细胞迁移的关键介质。它们通过与七种跨膜G蛋白偶联受体（GPCR）结合发挥作用，引起构象变化，从而触发参与细胞运动和受体活化的细胞内信号传导途径。尽管趋化因子被设计为执行发育和保护作用，但许多疾病是由这些蛋白质的不适当表达、调节或利用引起的。特定的趋化因子受体为HIV进入细胞提供了门户，而其他趋化因子受体则有助于炎症性疾病和许多类型癌症的迁移（转移）。因此，有相当大的兴趣，在描绘这些蛋白质的功能，以及拮抗其功能的机制的结构细节。目前有相当数量的信息的结构和受体结合表位的配体，这是小（8-12 kDa）的可溶性蛋白质。相比之下，关于它们的G蛋白偶联受体的结构信息知之甚少。这种信息的缺乏是由于在研究膜蛋白，特别是那些真核生物起源的固有的困难。表征GPCR的第一个障碍是不能获得足够水平的蛋白质，因为膜蛋白在过表达时通常是有毒的。通过晶体学或NMR对GPCR进行结构表征也比可溶性蛋白质困难得多。然而，经过十多年的配体结构-功能研究，我们最近开始关注受体，并有两个受体（D 6和CCR 1）的水平足以进行生物物理研究。在这个提议中，我们计划开发和应用氢氘交换质谱（DXMS）来表征趋化因子与其受体之间的结合界面。目的一是优化纯化受体的功能重建。在目标2中，我们将定义趋化因子配体上的受体结合表面。在目标3中，我们将开发方法来定义受体上的趋化因子结合表面。除了接触位点之外，还应该能够识别受体的胞内环区域，这些区域参与下游信号分子（如G蛋白）的激活，这些信号分子经历稳定性或构象的变化。如果成功的话，这些方法应该具有广泛的适用性，用于表征趋化因子：受体相互作用。例如，这些研究的结果可用于聚焦和补充诱变研究。由于CCR 1和D 6有许多不同的配体，因此最终可以研究不同的复合物;激动剂和拮抗剂的比较，特别是关于细胞内环的变化，将特别有趣。这些具体目标将通过两个小组的协同活动来实现：Dr. Handel，趋化因子和受体的结构生物学专家，Dr. Woods，DXMS方法学专家，他特别感兴趣，并且已经参与了开发膜蛋白的DXMS方法。公共卫生相关性：在正常生理条件下，趋化因子及其受体参与以它们在免疫系统功能和发育的背景下控制细胞迁移的能力为中心的过程。然而，不适当的趋化因子介导的细胞迁移和炎症引起或促成许多疾病的病理学，例如哮喘、类风湿性关节炎、多发性硬化症、心脏病和癌症。开发DXMS等方法来了解趋化因子的分子细节：受体相互作用和功能，可能有助于设计许多疾病的小分子和蛋白质治疗方法。

DESCRIPTION (provided by applicant): Chemokines are critical mediators of cell migration during immune surveillance, lymphocyte development, and inflammation. They function by binding to seven transmembrane G-protein coupled receptors (GPCRs), causing conformational changes that trigger intracellular signaling pathways involved in cell movement and receptor activation. Although chemokines were designed to carry out developmental and protective roles, many diseases result from inappropriate expression, regulation, or utilization of these proteins. Specific chemokine receptors provide the portals for HIV to get into cells, while others contribute to inflammatory disease, and migration (metastasis) of many types of cancers. Thus, there is considerable interest in delineating the structural details of how these proteins function, and mechanisms for antagonizing their function. Presently there is a fair amount of information on the structure and receptor binding epitopes of the ligands, which are small (8-12 kDa) soluble proteins. By contrast, very little structural information is known about their G protein-coupled receptors. This lack of information is due to the inherent difficulties in studying membrane proteins, particularly those of eukaryotic origin. The first obstacle in characterizing GPCRs has been the inability to obtain sufficient levels of protein because membrane proteins are usually toxic when over- expressed. Structural characterization of GPCRs by crystallography or NMR is also significantly more difficult than for soluble proteins. However, after more than a decade of structure-function studies of ligands, we recently began focusing on the receptors and have two receptors (D6 and CCR1) at levels sufficient for biophysical studies. In this proposal, we plan to develop and apply Hydrogen Deuterium Exchange coupled with Mass spectrometry (DXMS) to characterize the binding interfaces between chemokines and their receptors. In Aim 1, we will optimize the functional reconstitution of purified receptor. In Aim 2, we will define the receptor-binding surface on the chemokine ligand(s). In Aim 3, we will develop methods to define the chemokine-binding surface on the receptors. In addition to contact sites, it should be possible to identify regions of the intracellular loops of the receptor that are involved in activation of downstream signaling molecules like G proteins, which undergo changes in stability or conformation. If successful, the methods should have broad applicability for characterizing chemokine:receptor interactions. For example, the results of these studies can be used to focus and complement mutagenesis studies. Since there are many different ligands of CCR1 and D6, different complexes can ultimately be investigated; comparison of agonists and antagonists, especially with respect to changes in intracellular loops, will be particularly interesting. These specific aims will be accomplished by the synergistic activities of two groups: Dr. Handel, an expert in the structural biology of chemokines and receptors, and Dr. Woods, an expert in DXMS methodology, who is particularly interested, and already involved, in developing DXMS methods for membrane proteins. PUBLIC HEALTH RELEVANCE: Under normal physiological conditions, chemokines and their receptors are involved in processes centered around their ability to control cell migration in the context of immune system function and development. However, inappropriate chemokine-mediated cell migration and inflammation causes or contributes to the pathology of many diseases such as asthma, rheumatoid arthritis, multiple sclerosis, heart disease, and cancer. Developing methods like DXMS to understand the molecular details of chemokine:receptor interactions and function, may facilitate the design of small molecule and protein therapeutics for many diseases.