Structural basis of integral membrane enzyme function

完整膜酶功能的结构基础

• 批准号: 10609459
• 负责人: Filippo Mancia
• 金额: $ 43.35万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10609459
• 关键词: Active Sites; Alcohols; Anabolism; Animal Model; Attention; Biochemical; Biochemical Reaction; Biochemistry; Catalysis; Cell Wall; Cell physiology; Cells; Cellular Membrane; Cellular Structures; Charge; Complex; Coupling; Cryoelectron Microscopy; Data; Environment; Enzyme Interaction; Enzymes; Ethanolamines; Family; GDPmannose dolicholphosphate mannosyltransferase; Generations; Glycerophospholipids; Gram-Negative Bacteria; Hydrophobicity; Knowledge; Ligands; Ligase; Lipid A; Lipid Bilayers; Lipids; Lipopolysaccharides; Mediating; Membrane; Membrane Proteins; Modification; Molecular; O Antigens; Pathway interactions; Phase; Phosphotransferases; Play; Process; Production; Protein Glycosylation; Proteins; Reaction; Role; Signal Transduction; Specificity; Structure; Substrate Interaction; Substrate Specificity; System; Techniques; Transferase; X-Ray Crystallography; aqueous; chemical property; chemical reaction; chemical synthesis; enzyme structure; glycosylation; glycosyltransferase; human disease; hydrophilicity; lipophilicity; nanodisk; particle; protein function; protein-O-mannosyltransferase 1; reconstitution; structural biology; sugar; therapeutic target; tool

ABSTRACT Lipids are synthesized and modified primarily by integral membrane enzymes embedded, at least in part, in the bilayer itself. These enzymatic reactions are essential not only for the biosynthesis of all cellular membranes, but also for lipid-mediated signaling and for the export of soluble molecules as lipid conjugates to outer cellular compartments for a wide array of basic cellular functions, which include protein and lipid glycosylation, and modifications of the chemical properties of outer membranes as an adaptation of the cell to a changing environment. However, despite the advances in our understanding of how membrane proteins function, our knowledge of how membrane enzymes interact with their lipidic substrates at a molecular level has been scarce, also hindered by the hydrophobicity engendered by the lipid constituents themselves. The main focus of my lab is to use structural biology to investigate at a molecular level the interactions between membrane enzymes and their lipidic substrates. Our structures will produce testable functional hypotheses on how hydrophobic and hydrophilic substrates are brought into apposition for catalysis to occur, on how chemical reactions involving charged groups and an aqueous environment can adapt to process lipophilic molecules, on what are the molecular determinants of substrate specificity for hydrophobic ligands, and on the role that the membrane itself plays in these processes. We expect common principles on the interactions between, membrane, membrane enzymes, and lipidic substrates to emerge from our studies. We have focused our initial attention on glycerophospholipid biosynthesis as catalyzed by the CDP-alcohol phosphotransferase family of enzymes, and on the enzymatic coupling and uncoupling of sugars to polyisoprenyl carriers by the polyisoprenyl glycosyltransferase GtrB and the aminoarabinose transferase ArnT, respectively. We will continue in these directions by obtaining structures of these enzymes in complex with their lipidic ligands in mimics of the lipid bilayer environment, either by x-ray crystallography in lipidic cubic phase (LCP), or by single-particle cryo-electron microscopy (cryo-EM) in lipid-filled nanodiscs. We will also expand in new directions, related one with the other by the synthesis and modification of the lipopolysaccharide (LPS) component of Gram-negative bacteria (O-antigen ligase WaaL, ethanolamine transferase Ept A, and ArnT), by the coupling of activated sugars to polyisoprenyl carriers (GtrB and dolichol- phosphate mannose synthase, DPMS), and by the uncoupling of sugar-polyisoprenyl conjugates to generate mature LPS (WaaL), to modify lipid A (ArnT), or to glycosylate proteins (Pomt1/2). To succeed, we will combine our expertise in membrane protein production, biochemistry, and structural biology, to that of our collaborators that are leaders in their respective fields, ranging from chemical synthesis of sugar-lipid conjugates, to biochemical analysis of LPS, to functional analyses of membrane proteins in reconstituted systems or animal models, to the generation of tools to allow cryo-EM analysis of small proteins.

抽象的 脂质是合成和修饰的，主要是通过嵌入的整合性膜酶，至少部分地嵌入 双层本身。这些酶促反应不仅对于所有细胞膜的生物合成至关重要， 但也用于脂质介导的信号传导和用于脂质结合到外细胞的可溶性分子的导出 各种基本细胞功能的隔室，包括蛋白质和脂质糖基化，以及 外膜的化学特性的修饰作为细胞对变化的适应 环境。然而，尽管我们了解膜蛋白如何发挥作用，但我们的 了解膜酶在分子水平上如何与脂质底物相互作用的知识已经存在 稀缺，也受到脂质成分本身所产生的疏水性的阻碍。 我实验室的主要重点是使用结构生物学在分子水平上进行研究 膜酶及其脂质底物之间。我们的结构将产生可测试的功能 关于如何将疏水性和亲水性底物置于催化中的假设， 关于涉及带电组和水性环境的化学反应如何适应处理 亲脂性分子，关于疏水配体的底物特异性的分子决定因素是什么， 以及膜本身在这些过程中扮演的角色。我们期望关于 膜，膜酶和脂质底物之间的相互作用从我们的研究中出现。 我们将最初的注意力集中在甘油磷脂生物合成上，因为CDP-醇催化 酶的磷酸转移酶家族，以及糖的酶联和解偶联 聚异源载体通过聚异源糖基转移酶GTRB和氨基丁酰糖转移酶ARNT，ARNT 分别。我们将通过获得这些酶的结构与这些酶的结构进行复杂的结构来继续 它们的脂质配体在脂质双层环境的模仿中，要么通过脂质立方体中的X射线晶体学 相位（LCP）或通过脂质填充纳米盘的单粒子冷冻电子显微镜（冷冻-EM）。我们也会 通过新方向扩展，通过合成和修改将一个与另一个相关 革兰氏阴性细菌（O-抗原连接酶WAAL，乙醇胺）的脂多糖（LPS）成分 通过激活的糖与聚异源载体的偶联（GTRB和Dolichol-- 磷酸甘露糖合酶，dpms），通过糖 - 丙烯基借结合物的解偶联以产生 成熟的LPS（WAAL），以修饰脂质A（ARNT）或糖基化蛋白（POMT1/2）。 为了成功，我们将在膜蛋白质生产，生物化学和结构上结合我们的专业知识 生物学，我们的合作者是各自领域的领导者，从化学综合 糖脂偶联物的生化分析LPS，以对膜蛋白的功能分析 重建的系统或动物模型，以生成工具，以允许对小蛋白质进行冷冻分析。