Grant: Discovery Grants Program - Individual

The overall aim of this project is to determine how bacteria recognize and organize lipids in their outer membrane. The challenge we are tackling here is best described by Bruce Alberts, Editor of Science, who stated "I am painfully aware of the huge gap that remains in our understanding of even the simplest cells. Consider, for example, the common bacterium E. coli, which served as a predominant model organism in the early years of molecular biology. It is very sobering to report that more than 50 years later, nearly a quarter of the more than 4000 proteins encoded by its genome have functions that remain unknown. Might some new functional classes of biological molecules, common to all cells, be discovered by a focus on such proteins?"We will address this gap by elucidating the mechanism of an E. coli protein called YraP. There are at least 7000 proteins with similarity to YraP which have been found in a range of bacteria including various pathogens, and hence there are implications for infectious diseases. Moreover, as a unique gene product that is expressed in response to stress, YraP could offer opportunities for the design of antimicrobial agents once its structure, function and screening assays are available. The evolutionary relatives of YraP includes hemolysins, mechanosensitive channels, the membrane-pore forming protein Secretin, and a variety of eukaryotic proteins. We propose that they share a common function based on lipid recognition. Our focus is on understanding the cellular function, molecular interactions and 3D structures of YraP. We will reveal how this lipoprotein engages the phospholipids found in the membrane that surrounds all Gram negative bacteria. The outer membrane's role is to form a semi-permeable layer that controls the entry and exit of nutrients and other materials including drug molecules. The proteins inserted into this membrane include pores, channels and antigens that act as targets of immune responses. We propose that they all depend on YraP to deliver a specific lipid to the outer membrane. We will investigate how YraP recognizes and organizes this lipid, and how it traffics lipid from inside the cell. We will use methods including magnetic resonance spectroscopy to visualize the atoms of the structures and binding interfaces to expose the specific roles of its amino acid residues and lipids they contact. The resulting principles of ligand recognition and membrane biogenesis will inform the discovery of inhibitors, vaccines and antimicrobial agents. This project is collaborative, involving detailed structure-function analysis of a novel target by researchers at Canada's national NMR facility and the UK-based Institute of Microbiology and Infection. We will capitalize on our joint discovery of the role of YraP in lipid trafficking, along with a new NMR system geared to elucidate how this superfamily of proteins recognizes lipids, shapes membranes and protect cells.