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.
The atomic-level details of lipid/enzyme interactions and the determinants of their specificity remain poorly understood, despite their importance in biosynthesis and modification of cellular membranes, and in the assembly and use of lipid conjugates for essential cellular functions such as glycosylation. By applying structural biology techniques to enzymes that are involved in membrane biosynthesis, assembly and modification of the lipopolysaccharide component of the cell wall of Gram-negative bacteria, and processes of protein glycosylation, we will gain testable hypotheses on the basic principles of substrate recognition, and on how membrane, membrane enzymes, and lipidic substrates interact to facilitate these reactions. These data should also reveal the molecular mechanisms of select human diseases, and provide pathways toward new classes of therapeutics targeting lipid/enzyme active sites.
