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Structural basis of integral membrane enzyme function

完整膜酶功能的结构基础

基本信息

批准号：
9921455
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
项目类别：
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/9921455
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 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

项目摘要

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