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

突触结合蛋白样蛋白的钙非依赖性和钙抑制性膜结合 项目总结/摘要 蛋白质-膜相互作用是囊泡运输和胞吐中大多数重要步骤的基础， 包括胰岛素等激素。虽然许多关键蛋白质是已知的， 膜相互作用的结构机制的研究对于未来选择性抑制剂的设计是必要的。 本研究的主要目标是提供突触结合蛋白的机制和结构理解， Like蛋白质（Slp），其抑制可能在药学上有用的关键蛋白质家族。两 将研究突触结合蛋白样蛋白（Slp）家族的代表性且独特的成员： Slp-2和Slp-4。这些被选为我们的重点，因为（i）膜结合的结构 Slp-4的C2 A结构域是已知的，并且（ii）Slp-2表现出有趣的和不寻常的Ca 2+抑制特性， 膜结合通常，Slp蛋白的功能是在分泌囊泡和血浆之间架起桥梁 膜，稳定对接分泌囊泡胞吐作用前。Slp蛋白具有N-末端结构域 结合分泌囊泡上的Rab GTP酶，和一个或两个C-末端C2结构域，其与高 通过与阴离子脂质分子如磷脂酰肌醇-（4，5）- 二磷酸（PIP 2）和磷脂酰丝氨酸（PS）。大多数Slp蛋白，包括Slp-4， 然而，Slp-2的膜亲和力被钙唯一地抑制。这 该项目是基于这样的假设，即高亲和力Slp C2 A-膜相互作用涉及多重结合 脂质和/或其他配体的位点，其中之一起别构作用，是钙抑制的基础， Slp-2具体目标1问：“Slp-4 C2 A结构域的哪些残基是高亲和力膜结合所需的？ 对接？”这个问题将使用实验和计算方法相结合来回答 基于该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以与膜脂质相互作用。网站的影响- 定向诱变将使用定量脂质体结合测定和细胞- 基于分泌的模型系统。根据以前的报道，预测基本上 相对于野生型蛋白，减弱的膜结合将增强胰岛素分泌。具体目标2问， “在突触结合蛋白样蛋白中保守的口袋会抑制脂质结合吗？”为了这个目标， 钙抑制Slp-2C 2A的结构机制将使用基于荧光的脂质来探测， 钙结合测量和电子顺磁共振。我们的目标是发展一个结构性的 一个位点的钙结合如何抑制远端位点的膜结合。如果钙结合到 Slp-2 C2 A结构域诱导阻断膜结合的结构变化，然后相应的 Slp-4的残余口袋将是发现抑制性化合物的潜在靶点。因为Slp-4限制了 在体内的胰岛素分泌中，其抑制代表了未来糖尿病治疗的可能途径。

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.