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.

摘要 脂质的合成和修饰主要是由至少部分地嵌入在膜中的膜酶来完成的。 bilayer本身这些酶促反应不仅对于所有细胞膜的生物合成是必需的， 而且还用于脂质介导的信号传导和用于将可溶性分子作为脂质缀合物输出到外细胞 用于广泛的基本细胞功能的区室，包括蛋白质和脂质糖基化，以及 外膜的化学性质的改变，作为细胞对变化的环境的适应。 环境然而，尽管我们对膜蛋白功能的理解有了进步， 关于膜酶如何在分子水平上与它们的底物相互作用的知识， 稀缺，也受到脂质成分本身产生的疏水性的阻碍。 我实验室的主要重点是利用结构生物学在分子水平上研究 在膜酶和它们的底物之间。我们的结构将产生可测试的功能 关于疏水性和亲水性基质如何并置以发生催化作用的假设， 涉及带电基团和水环境的化学反应如何适应过程 亲脂性分子，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所起的作用。我们期待共同的原则， 之间的相互作用，膜，膜酶，和生物底物从我们的研究中出现。 我们最初的注意力集中在CDP-醇催化的甘油磷脂生物合成上 磷酸转移酶家族的酶，以及糖的酶促偶联和解偶联， 通过聚异戊二烯基糖基转移酶GtrB和氨基阿拉伯糖转移酶ArnT将聚异戊二烯基载体， 分别我们将通过获得这些酶的结构来继续这些方向， 在模拟脂质双层环境中，通过X射线晶体学在双折射立方晶体中， 相（LCP），或通过脂质填充的纳米盘中的单颗粒低温电子显微镜（cryo-EM）。我们还将 在新的方向上扩展，通过合成和修改彼此相关， 革兰氏阴性菌的脂多糖（LPS）组分（O-抗原连接酶WaaL，乙醇胺 转移酶Ept A和ArnT），通过将活化的糖偶联到聚异戊二烯基载体（GtrB和多萜醇- 磷酸甘露糖合酶，DPMS），并通过糖-聚异戊二烯基缀合物的解偶联， 成熟LPS（WaaL），修饰脂质A（ArnT），或糖基化蛋白（Pomt 1/2）。 为了取得成功，我们将联合收割机结合我们在膜蛋白生产、生物化学和结构方面的专业知识， 生物学，我们的合作者是各自领域的领导者，从化学合成 的糖-脂质缀合物，LPS的生物化学分析，细胞膜蛋白的功能分析， 重组系统或动物模型，以产生工具，允许冷冻EM分析小蛋白质。