Structure and function of novel G protein conformations

新型G蛋白构象的结构和功能

• 批准号: 9532410
• 负责人: Sharon L Campbell
• 金额: $ 2.18万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9532410
• 关键词: Adopted; Affect; Bacterial Toxins; Binding; Biochemistry; Biological; Biophysics; Cardiovascular system; Cell Surface Receptors; Cell surface; Chemicals; Cholera; Circular Dichroism; Collaborations; Communicable Diseases; Data; Defect; Diarrhea; Disease; Epidemic; Fluorescence; G-Protein-Coupled Receptors; G-protein Beta gamma; G-substrate; GTP Binding; GTP-Binding Protein alpha Subunits; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; GTPase-Activating Proteins; Genetic; Goals; Guanine Nucleotides; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Haiti; Heart Diseases; Human; Human Pathology; Label; Laboratories; Lead; Malignant Neoplasms; Methods; Molecular Conformation; Molecular Genetics; Mutation; Myocardial Ischemia; Nucleotides; Nutrient; Ocular Melanoma; Oncogenic; Pertussis; Pharmacologic Substance; Pharmacology; Population; Protein Conformation; Protein Kinase; Protein phosphatase; Proteins; Receptor Activation; Regulation; Reperfusion Injury; Research; Resolution; Sensory; Signal Transduction; Signaling Protein; Site; Stress; Stroke; Structure; Suppressor Mutations; Techniques; Testing; Time; Variant; Work; X-Ray Crystallography; biophysical analysis; design; disease-causing mutation; human disease; inhibitor/antagonist; innovation; killings; mutant; novel; prevent; protein function; protein protein interaction; protein structure; public health relevance; receptor; reconstitution; small molecule; small molecule inhibitor; small molecule therapeutics; targeted treatment; ubiquitin ligase

 DESCRIPTION (provided by applicant): Many biological signals act via cell surface receptors and GTP-binding G proteins. Recent findings have revealed new and unexpected conformational changes in the G protein α subunit. Our overall hypothesis is that G proteins exist in a larger ensemble of functionally-distinct structural conformations, some of which may be targeted therapeutically. We propose to investigate these changes and their consequences for signaling. The project is a collaboration between two laboratories with distinct and complementary expertise, and will combine molecular genetics (Dohlman), structural biophysics (Campbell), biochemistry and pharmacology. The proposed work is innovative because it employs, for the first time, high resolution NMR to investigate Gα protein conformations and inhibitor binding interactions. The proposed work is significant because perturbations in G protein function underlie human pathologies including cardiovascular damage, infectious disease, and cancer. Aim 1. Functional analysis of Gα proteins in human diseases (Dohlman). Several human cancers arise from mutations in Gα. Cholera is an infectious and deadly form of diarrhea, caused by a bacterial toxin that covalently modifies Gαs. In these instances the G protein is no longer able to hydrolyze GTP. We have identified a panel of second-site suppressor mutations that appear to stabilize the inactive (GDP-like) conformation, despite an inability to hydrolyze GTP. We will now compare the function of G proteins with GTPase deficient (oncogenic) mutations or that are ADP-ribosylated, in the absence or presence of our suppressor mutations. We will reconstitute Gα with its known binding partners and determine how these suppressor mutations affect interactions with guanine nucleotides, G protein βγ subunits, the GTPase- activating protein, as well as recently identified protein kinases and phosphatases. Aim 2. Structural analysis of Gα proteins in human diseases (Campbell). Gα proteins undergo dramatic conformational changes during activation and inactivation. These changes have so far been documented by low resolution or static techniques. Our new preliminary data show the feasibility of conducting high resolution NMR on Gα proteins. NMR is ideal for detecting structural and dynamic changes in proteins, including those that are transient and dynamic. We will now conduct a detailed analysis of the conformational changes imposed by mutational activation, in the absence or presence of our suppressor mutations, and in the absence or presence of small molecules that bind to Gα. In the longer term, this work will reveal the structural basis for altered protein-protein interactions as well as the potential for small molecule suppressors of disease- causing mutations.

 描述（由申请人提供）：许多生物信号通过细胞表面受体和GTP结合G蛋白发挥作用。最近的研究结果揭示了G蛋白α亚基中新的和意想不到的构象变化。我们的总体假设是，G蛋白存在于一个更大的功能不同的结构构象，其中一些可能是有针对性的治疗。我们建议调查这些变化及其对信号的影响。该项目是两个具有独特且互补专业知识的实验室之间的合作，并将联合收割机分子遗传学（Dohlman）、结构生物物理学（坎贝尔）、生物化学和药理学结合起来。这项工作是创新的，因为它首次采用高分辨率NMR来研究Gα蛋白的构象和抑制剂结合的相互作用。这项工作意义重大，因为G蛋白功能的扰动是人类病理学的基础，包括心血管损伤、传染病和癌症。 目标1。Gα蛋白在人类疾病中的功能分析（Dohlman）。几种人类癌症是由Gα突变引起的。霍乱是一种传染性和致命的腹泻形式，由共价修饰Gα的细菌毒素引起。在这些情况下，G蛋白不再能够水解GTP。我们已经确定了一组第二个网站的抑制突变，似乎稳定无活性（GDP样）的构象，尽管无法水解GTP。我们现在将比较G蛋白与GT3缺陷（致癌）突变或ADP-核糖基化，在我们的抑制突变的存在或不存在的功能。我们将重组Gα与其已知的结合伙伴，并确定这些抑制突变如何影响与鸟嘌呤核苷酸，G蛋白βγ亚基，GT3活化蛋白，以及最近发现的蛋白激酶和磷酸酶的相互作用。 目标二。人类疾病中Gα蛋白的结构分析（坎贝尔）。Gα蛋白在活化和失活过程中经历剧烈的构象变化。迄今为止，这些变化都是通过低分辨率或静态技术记录的。我们的新的初步数据显示了对Gα蛋白进行高分辨率NMR的可行性。NMR是检测蛋白质结构和动态变化的理想工具，包括那些瞬时和动态变化。我们现在将对突变激活引起的构象变化进行详细的分析，在不存在或存在我们的抑制突变的情况下，以及在不存在或存在结合Gα的小分子的情况下。从长远来看，这项工作将揭示改变蛋白质-蛋白质相互作用的结构基础以及致病突变的小分子抑制剂的潜力。