Structural basis of phosphatidylglycerol recognition and trafficking at the outer membrane

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The overall aim of this project is to determine how bacteria recognize and organize lipids in their outer membraneThe challenge we are tackling here is best described by Bruce Albert, the Editor of Science, who recently 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?" [Science, 2012, 337: 6102]. We intend to fill this gap of biological knowledge by defining the function and mechanism of an E.coli coli protein called YraP. According to the Interpro database there are at least 6746 proteins with similarity to YraP which have been sequenced from a wide range of bacterial species including many pathogens, and hence there is broader relevance to many micro-organisms and infectious diseases. Moreover, as a unique bacterial gene that is expressed in response to stress, YraP offers potential opportunities for the design of antimicrobial agents, especially once suitable screening assays and mechanistic insights are available. The diversity of YraP-related proteins, which typically are composed of a pair of BON domains, includes haemolysins, mechanosensitive channels, the membrane-pore forming protein Secretin, and several eukaryotic proteins. This suggests a common function involving lipid recognition or membrane manipulation, as first proposed by C Yeats and A Bateman in 2003, although how they are related at a structural and mechanistic level remains unclear.Our primary focus is on solving the atomic resolution structures and molecular interactions of YraP, providing the first experimental insights into how the BON domain engages the phospholipid components of the protective outer membrane that surrounds all Gram negative bacteria. The outer membrane's main function is to form a semi-permeable layer that controls the influx and efflux of nutrients and other materials including drug molecules. The proteins inserted into outer membranes include 90 lipid modified proteins like YraP as well as pores, channels and antigens that act as targets of immune responses. These lipoproteins are embedded into a bilayer composed of immunogenic lipopolysaccharides, phospholipids and glycolipids. We will investigate how YraP binds and organizes lipids and contributes to trafficking them to the outer membrane of bacteria. In order to develop the first mechanistic understanding of YraP function, we will use biophysical methods including nuclear magnetic resonance spectroscopy (NMR). Visualising the atoms of the protein structures and ligand interactions of YraP will provide valuable insights into the functional roles of the amino acid residues and lipids they contact. These principles of PG recognition and outer membrane specificity could aid in the discovery of molecular inhibitors and new classes of antimicrobial agents, with YraP playing a key role in controlling cell permeability and ensuring viability during bacterial stress.This project represents collaborative science, involving a joint structure-function analysis of a novel target by two research groups from the Institute of Microbiology and Infection and the Henry Wellcome Building for Biomolecular NMR Spectroscopy (HWB-NMR), respectively. We will combine the molecular and cellular insights of Ian Henderson's bacteriology group, which has just discovered the role of YraP in lipid trafficking, along with the structural biological expertise within Michael Overduin's group, which specialises in elucidating protein:lipid recognition and trafficking mechanisms.