Structural basis of phosphatidylglycerol recognition and trafficking at the outer membrane

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 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.

In this project we aim to:

1. To elucidate the three dimensional structures of the multiple states of the YraP lipoprotein which we have discovered to bind and potentially regulate phospholipids, characterising its unique fold and binding surfaces. The solution structures will be contrasted with other bacterial homologues in order to determine whether there are specialized functional features including electrostatic and hydrophobic surfaces and druggable sites in this accessible target.

2. To measure the binding properties of the YraP protein for lipid and protein components of the outer membrane to systematically define its biological interaction network at a quantitative level. The interaction sites will be mapped and mutated for analysis of specific effects on ligand binding properties and for prediction of the functions of diverse BON superfamily members.

3. To determine which outer membrane lipids interact with the YraP lipoprotein in order to define the specific electrostatic and hydrophobic contacts responsible for their insertion, orientation and assembly on the membrane. The structures of micelle, bicelle and lipid disc complexes will be solved, allowing realistic models to be calculated of how YraP recognizes ligands, inserts into bilayers and alters membrane properties.

4. The in vivo function of YraP will be elucidated based on the structural and functional roles of individual residues which bind lipids, insert into micelles or contact outer membrane proteins. These roles will be tested by fluorescence microscopy and co-immunoprecipitations to detect co-localisation and distributions of outer membrane components in E coli cells expressing mutant and wild type YraP and its partners.

Together this will provide the first experimental insights into the biochemical function and 3D structure of a double BON domain protein, and will illuminate the lipid trafficking pathway from the inner membrane to the outer membrane of Gram negative bacteria.

Broader Academic Community: Scientific groups will be engaged by primary journal articles, reviews, web sites, exchange visits and access to research products and services. Overduin, Henderson and Knowles have produced over 50 manuscripts since 2008, including papers in JACS, EMBO R, Nature Str Mol Biol, PLOS, PNAS USA and Science. Open access journals will continue to be the medium for communicating our results. Overduin operates websites at www.nmr.bham.ac.uk as well as HWB-NMR twitter and facebook accounts to disseminate new knowledge, practices and events. The team will continue to presents at and organise conferences, and will run focused meetings will be used to transfer knowledge and skills, with Overduin being the organizer of four BBSRC-funded JPA workshops on protein expression, annual EU-funded meetings on membrane-protein interactions, and Wellcome Trust-funded NMR workshops on NMR software and data analysis.

Commercial Sector: Pharmaceutical and biotech companies will learn about the tools and methods being developed here through their visits to Birmingham as HWB-NMR users, as well as through collaborations, with GSK and AstraZeneca supporting BBSRC CASE PhD studentships on academic research in Overduin's group. Overduin, Knowles and Henderson have been closely involved in the BBSRC BRIC programme, which brings together industry leaders in the bioprocessing sector, in order to develop and share new technologies for membrane protein production, genetic engineering of E. coli and cell based screening assays.

Wider Public: The PIs will continue to play an active role in promoting the public understanding of science, and have contributed to stories in the Birmingham Post, Business Desk, Guardian, Information Daily, Telegraph, BBC-WM and Research TV in the past several years. Overduin is a member of the steering group of the British Science Association, which held a Science Festival in Birmingham in Sept 2010, and every four years thereafter, and runs events for this and University Open Days. Lab and facility tours will continue to given by Overduin and members of his group to high school classes and the public, and several high school students have performed summer research projects in his lab recently through Nuffield bursories.

Overduin also volunteers as Chair of the Science and Medicine Forum of the Lunar Society, a scientific body which was originally founded in the West Midlands in 1775. He serves on its Executive Committee, helping to organize monthly lectures and public events with attendances of up to 600 people. Speakers he has hosted include the Nobel Laureate Paul Nurse, President, Rockefeller University and Sir Liam Donaldson, Chief Medical Officer. This provides an avenue to present results on pharmaceutical research and genetics, proteomics and systems biology of disease, helping to overcome public anxiety about pathogenic diseases, and demonstrating the benefits of new technologies and drug discovery through academic - industrial collaborations.

Business engagement: Overduin has served as a member of the University of Birmingham's Regional Advisory Group which is tasked with exploring strategic collaborations with regional partners. He established Science Capital as a nonprofit organization to connect internationally recognized scientists with the public and businesses to present and discuss innovations and priorities for growth and investment. For example, he presented his BBSRC-funded research alongside AstraZeneca and Sygnature Discovery to an audience of 100 such experts on 19 Sept 2012 in order to provide awareness of opportunities including open innovation-based drug discovery.

The cost of these public engagement activities are estimated at 3 hours per month, as well as the associated local travel costs (~£20/month), all of which is given freely for this publicly funded research.