Characterization of CCR5 Receptor N-terminal domain

描述(由申请人提供)：趋化因子受体CCR5和CXCR4与CD4和病毒包膜糖蛋白(Env)一起，介导人类免疫缺陷病毒1型(HIV-1)的感染。我们的长期目标是了解趋化因子受体的结构特征，使它们能够作为进入HIV-1的门户。这些结构知识将为设计受体诱饵作为艾滋病毒相关疾病的治疗药物提供有价值的见解。目前的鸡尾酒疗法大幅降低了死亡率，但病毒可能会发生突变，并对现有的抑制病毒蛋白功能的药物产生抗药性。因此，靶向宿主蛋白，如CCR5趋化因子受体，提供了一个有吸引力的替代策略。CCR5受体N-末端结构域(N-结构域)上的一簇带电氨基酸和硫化酪氨酸在HIV结合中起着关键作用。酪氨酸硫化N结构域的多肽竞争性地抑制HIV与受体的结合，并促进HIV与缺乏N结构域的突变受体的结合。我们的初步数据显示，受体N-结构域在溶液中是非结构化的，但在模拟“天然膜环境”的洗涤剂胶束中有结构。这些观察结果表明，受体N-结构域作为一个自主折叠的结构和功能单位发挥作用。这使得研究分离的受体N-结构域可以深入了解天然结构域的结构和灵活性，以及结合诱导的结构和动态变化。核磁共振波谱非常适合于这类研究，但需要大量(Mg)重组蛋白，最好是同位素标记的(13C和15N)和酪氨酸硫化。在目标1中，我们将在大肠杆菌和昆虫细胞中表达CCR5 N结构域，并优化其高效生产、纯化、溶解和硫酸盐化的条件。在目标2中，我们将使用核磁共振和其他生物物理技术来表征自由和配体结合的CCR5 N结构域的结构。我们将使用体外试验来测试受体域肽抑制HIV感染的能力。我们的方法是新颖的，成功实现我们的目标对于理解CCR5在艾滋病毒感染中的结构作用至关重要。最重要的是，根据CCR5 N结构域结构设计的受体诱骗应该会产生预防/治疗艾滋病毒相关疾病的药物。

DESCRIPTION (provided by applicant): The chemokine receptors CCR5 and CXCR4, in conjunction with CD4 and the viral envelope glycoprotein (Env), mediate infection by human immunodeficiency virus type 1 (HIV-1). Our long-term objective is to understand the structural features of chemokine receptors that enable them to function as portals for HIV-1 entry. Such structural knowledge will provide valuable insight for designing receptor decoys as therapeutics for HIV-related diseases. The current cocktail treatment has dramatically reduced mortality, but the viruses can mutate and become resistant to the existing drugs that inhibit viral protein function. Therefore, targeting host proteins such as the CCR5 chemokine receptor provides an attractive alternative strategy. A cluster of charged amino acids and sulfated tyrosines in the CCR5 receptor N-terminal domain (N-domain) play a critical role in HIV binding. Tyrosine-sulfated N-domain peptides competitively inhibit HIV binding to the receptor, and facilitate HIV binding to a mutant receptor lacking the N-domain. Our preliminary data show that the receptor N-domains are unstructured in solution, but structured in detergent micelles that mimic the " native membrane environment. These observations suggest that the receptor N-domain functions as an autonomous folded structural and functional unit. This allows studying isolated receptor N-domain to gain insight into the native domain's structure and flexibility, and binding-induced structural and dynamic changes. Nuclear magnetic resonance (NMR) spectroscopy is ideally suited for such studies, but requires large (mg) quantities of recombinant protein, preferably isotopically labeled (13C and 15N) and tyrosine-sulfated. In aim 1, we will express the CCR5 N-domain both in E.coli and insect cells, and optimize conditions for its high-level production, purification, solubility, and sulfation. In aim 2, we will use NMR and other biophysical techniques to characterize the structure of the free and ligand-bound CCR5 N-domain. We will use an in vitro assay to test the receptor domain peptides ability to inhibit HIV infection. Our approaches are novel, and successful accomplishment of our aims is critical for understanding the structural role of CCR5 in HIV infection. Most importantly, receptor decoys designed on the basis of CCR5 N-domain structure should result in drugs for preventing/treating HIV-related diseases.