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Structural basis of the outer membrane protein assembly system by NMR spectroscopy

Reference	BB/G022054/1
Principal Investigator / Supervisor	Professor Michael Overduin
Co-Investigators / Co-Supervisors	Professor Ian Henderson
Institution	University of Birmingham
Department	Cancer Sciences
Funding type	Research
Value (£)	540,064
Status	Completed
Type	Research Grant
Start date	01/12/2009
End date	30/11/2013
Duration	48 months

Abstract

We aim to: 1. To elucidate the three dimensional structures of at least three of the NlpB, SpmA, YfgL and YfiO lipoproteins which form core components of the BAM complex, characterizing their unique binding surfaces. The structures of these proteins, which range in size from 12.3 to 41.8 kDa and yield excellent NMR spectra, will be contrasted with other bacterial homologues and human proteins in order to determine whether there are speciallized functional features and druggable pockets in these essential and accessible antimicrobial targets. 2. To measure the protein binding properties of each BAM component to systematically define the interaction network at a quantitative level. The protein interactions within this dynamic assembly machine will be mapped and complexes identified by surface plasmon resonance (SPR) and analytical ultracentrifugation (AUC) to enable NMR and SAXS analysis of complexed structures. Interactions with periplasmic chaperones, which deliver proteins for folding, will be investigated similarly in order to build testable models of the transfer event. Residues involved in intermolecular interactions will be mutated in order to inhibit and stabilize the assembly complexes, providing a basis for reconstruction of the assembly and folding process. 3. To determine how outer membrane lipids interact with the lipoproteins in order to define the specific electrostatic and hydrophobic contacts responsible for their insertion, orientation and assembly on the membrane. The structures of micelle complexes will be calculated by our HADDOCK approach using paramagnetic resonance effects and intermolecular NOEs, allowing models to be constructed of how the BAM components and complexes interface with the outer membrane surface. The residues which insert into the membrane and mediate specific lipid interactions will be tested by NMR and SPR studies of mutants forms, which our collaborator Henderson will also validate by independent in vivo assays.

Summary

In this research project, the three dimensional structures and functions of proteins found in the membrane that surrounds bacterial cells will be analyzed by biophysical methods including magnetic resonance spectroscopy. We will characterize the molecular mechanisms of four Escherichia coli proteins which interact with the YaeT transmembrane protein. This bacterial protein complex spans the outer membrane and interacts with other proteins from the periplasmic space, secreting them out of the cell and into the outer membrane and surrounding environment. Here we focus on four accessory lipoproteins, NlpB, YfgL, YfiO and SmpA, which form an essential complex with YaeT and are found in essentially all Gram negative bacteria. Together they recruit and fold all outer membrane proteins which form barrel folds. Gram negative bacteria are characterized by a protective outer membrane that surrounds a polymeric network known as peptidoglycan. The outer membrane's main function is to form a semi-permeable layer around the peptidoglycan. For example, it controls the influx and efflux of nutrients and other materials including drug molecules. Proteins including pores and channels are inserted into the outer membrane in order to regulate its permeability. Some of the proteins inserted into the outer membrane are important antigens that act as targets of protective immune responses e.g. the autotransporter proteins, the most widely used protein secretion system within all Gram-negative bacteria. The analysis of many bacterial genomes has revealed that the conserved protein assembly complex we are studying is universally found in outer membranes of Gram negative bacteria, and is essential for their survival. This basic system is also found in some Gram positive bacteria and mitochondria and chloroplasts, reflecting the bacterial origins of such organelles, where it is also essential for survival. Thus, although we are focussing on the E coli system, the results of our work will have broad implications for protein assembly and folding in a diverse range of cell types and organelles. The lipoproteins we are studying are responsible for the production of outer membrane proteins in their folded and functional forms, and hence are important for the viability and normal physiology of the bacterial cell. Understanding how the four lipoproteins recognize and assemble proteins is important for targeting pathogenic bacteria and for manipulating the immune response during a bacterial infection. Visuallizing their structures and characterizing their ligand interactions and binding pockets provides valuable mechanistic insights that could aid in the discovery of molecular inhibitors and new classes of antimicrobial agents. The outer membranes of different types of Gram-negative bacteria contain a variety of lipids in addition to proteins. The outer leaflet is composed of lipopolysaccharides, glycolipids and phospholipids which differ between bacterial species. The lipids in the outer membrane are essential for the assembly of proteins into the membrane, and also have important roles in immune response. Consequently, we will also investigate the interactions of lipids and membranes with lipoproteins in order to better understand how the lipoproteins are oriented and assemble at the membrane surface.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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