Ca2+-independent and Ca2+-inhibited membrane binding by synaptotagmin-like proteins

突触结合蛋白样蛋白的 Ca2 不依赖性和 Ca2 抑制膜结合

• 批准号: 9496381
• 负责人: Jefferson D. Knight
• 金额: $ 3.94万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9496381
• 关键词: Affinity; Allosteric Site; Beta Cell; Binding; Binding Sites; Biological Assay; Biological Models; Biomedical Research; C-terminal; C2 Domain; Calcium; Calcium Binding; Calcium ion; Cations; Cell membrane; Cells; Cellular biology; Charge; Closure by clamp; Collaborations; Colorado; Computer Simulation; Data; Development; Diabetes Mellitus; Distal; Docking; Electron Spin Resonance Spectroscopy; Electrostatics; Exhibits; Exocytosis; Fluorescence; Foundations; Future; Glucagon; Goals; Homologous Gene; Hormones; In Vitro; Insulin; Ions; Kinetics; Ligand Binding; Ligands; Lipid Binding; Lipids; Liposomes; Measurement; Measures; Membrane; Membrane Lipids; Membrane Proteins; Methods; Modeling; Molecular Conformation; Molecular Structure; Mutation; N-terminal; Pancreatic Hormones; Pathway interactions; Pharmacologic Substance; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphatidylserines; Physical Chemistry; Population; Property; Protein Family; Protein Region; Proteins; Records; Reporting; Secretory Vesicles; Site; Site-Directed Mutagenesis; Structural Models; Structure; Structure-Activity Relationship; Students; Surface; Techniques; Tertiary Protein Structure; Testing; Training; Universities; Vesicle; Work; analog; base; computational chemistry; design; diabetes mellitus therapy; experimental study; granuphilin; in vivo; inhibitor/antagonist; insight; insulin secretion; knock-down; member; molecular dynamics; mutant; overexpression; protein function; rab GTP-Binding Proteins; synaptotagmin; trafficking; undergraduate student

Ca2+-independent and Ca2+-inhibited membrane binding by synaptotagmin-like proteins Project Summary/Abstract Protein-membrane interactions underlie most of the important steps in vesicle trafficking and exocytosis, including for hormones such as insulin. Although many of the key proteins are known, a better understanding of the structural mechanisms of membrane interaction is necessary for the future design of selective inhibitors. The broad goal of this study is to provide such a mechanistic and structural understanding for synaptotagmin- like proteins (Slp), a key family of proteins whose inhibition may be pharmaceutically useful. Two representative and yet unique members of the synaptotagmin-like protein (Slp) family will be investigated: Slp-2 and Slp-4. These have been chosen as our focus because (i) the structure of the membrane-binding C2A domain from Slp-4 is known, and (ii) Slp-2 exhibits an intriguing and unusual property of Ca2+-inhibited membrane binding. Generally, Slp proteins function to bridge between secretory vesicles and the plasma membrane, stably docking secretory vesicles prior to exocytosis. Slp proteins possess an N-terminal domain that bind Rab GTPases on secretory vesicles, and one or two C-terminal C2 domains that dock with high affinity to plasma membranes via interaction with anionic lipid molecules such as phosphatidylinositol-(4,5)- bisphosphate (PIP2) and phosphatidylserine (PS). Most Slp proteins, including Slp-4, bind membranes independently of calcium ions; however, the membrane affinity of Slp-2 is uniquely inhibited by calcium. This project is based on the hypothesis that high-affinity Slp C2A–membrane interaction involves multiple binding sites for lipids and/or other ligands, one of which acts allosterically and is the basis for calcium inhibition in Slp-2. Specific Aim 1 asks, “Which residues of the Slp-4 C2A domain are required for high-affinity membrane docking?” This question will be answered using a combination of experimental and computational approaches based on the known molecular structure of this protein domain. Molecular dynamics simulations will predict how different regions of the protein domain act together to interact with membrane lipids. Effects of site- directed mutagenesis will be tested experimentally using both quantitative liposome binding assays and cell- based model systems of secretion. Based on previous reports, it is predicted that mutants which substantially weaken membrane binding will enhance insulin secretion relative to the wild-type protein. Specific Aim 2 asks, “Does a pocket conserved among synaptotagmin-like proteins inhibit lipid binding allosterically?” For this Aim, the structural mechanism of Slp-2 C2A inhibition by calcium will be probed using fluorescence-based lipid and calcium binding measurements and electron paramagnetic resonance. The goal is to develop a structural model for how calcium binding at one site inhibits membrane binding at a distal site. If calcium binding to the Slp-2 C2A domain induces a structural change that blocks membrane association, then the corresponding vestigial pocket in Slp-4 would be a potential target for discovering inhibitory compounds. Because Slp-4 limits insulin secretion in vivo, its inhibition represents a possible avenue for future diabetes therapy.

突触素样蛋白对钙离子非依赖性和钙离子抑制的膜结合 项目摘要/摘要 蛋白-膜相互作用是囊泡运输和胞吐过程中最重要的步骤。 包括胰岛素等荷尔蒙。虽然许多关键蛋白质是已知的，但更好地理解 了解膜相互作用的结构机制对于未来选择性抑制剂的设计是必要的。 这项研究的广泛目标是为突触聚集蛋白提供这样一种机制和结构上的理解。 像蛋白质(SLP)一样，是一个关键的蛋白质家族，其抑制作用可能在药学上有用。二 突触素样蛋白(SLP)家族中具有代表性但又独一无二的成员将被研究： SLP-2和SLP-4。之所以选择这些作为我们的重点，是因为(I)膜结合的结构 SLP-4的C2a结构域是已知的，并且(Ii)SLP-2表现出一种耐人寻味的和不寻常的钙抑制特性 膜结合。一般来说，SLP蛋白在分泌囊泡和血浆之间起桥梁作用。 膜，在胞吐之前稳定地对接分泌小泡。SLP蛋白具有N-末端结构域 将Rab GTP酶结合在分泌小泡上，以及一个或两个C-末端C2结构域，与High 通过与磷脂酰肌醇-(4，5)-阴离子脂分子相互作用而与质膜亲和力 二磷酸(PIP2)和磷脂酰丝氨酸(PS)。大多数SLP蛋白，包括SLP-4，都与膜结合 不依赖于钙离子；然而，SLP-2的膜亲和力唯一地被钙抑制。这 该项目基于高亲和力SLP C2a-膜相互作用涉及多个结合的假设 脂类和/或其他配体的位点，其中一种是变构作用的，是钙抑制的基础。 SLP-2。特定目的1问：高亲和力膜需要SLP-4C2a结构域的哪些残基 对接？“这个问题将用实验和计算相结合的方法来回答。 基于这个蛋白质结构域的已知分子结构。分子动力学模拟将预测 蛋白质结构域的不同区域如何共同作用于膜脂类。场地的影响- 定向诱变将在实验中使用定量脂质体结合分析和细胞- 基于分泌物的模型系统。根据以前的报道，预测突变体基本上是 相对于野生型蛋白，膜结合减弱会增强胰岛素的分泌。《特定目标2》要求， “在突触素样蛋白中保守的口袋是否会抑制变构作用下的脂质结合？”为了达到这个目的， 钙抑制SLP-2C2a的结构机制将用基于荧光的脂质和 钙结合测量和电子顺磁共振。目标是开发一种结构性的 一个部位的钙结合如何抑制远端部位的膜结合的模型。如果钙结合到 SLP-2C2a结构域导致结构改变，阻断膜结合，然后相应的 SLP-4中的残留口袋可能是发现抑制性化合物的潜在靶点。因为SLP-4限制了 体内的胰岛素分泌，其抑制代表了未来糖尿病治疗的一条可能途径。