How do ATP-independent chaperones assist OMP folding and assembly? Insights from mass spectrometry and other approaches

Every living cell is surrounded by an envelope, called a membrane, which acts as a barrier to the external environment. These membranes are comprised of lipids and proteins that regulate the entry and exit of molecules in to and out of the cell: in particular regulating the entry of nutrients that ensure that the organism is able to grow and survive. Some bacteria, called gram-negative bacteria, are surrounded by two membranes, one inside the other, called the inner and outer membranes, with a cellular compartment in-between called the periplasm. The outer membrane (OM) of such bacteria contains a distinct selection of lipids, and is crowded with OM proteins (OMPs) that are vital for bacterial survival. New OMPs are made by the bacteria all the time, but the machinery that is used to make these proteins is located in the main compartment of the cell (the cytoplasm), inside the inner membrane. Once made, the OMPs have to undertake a long journey to where they are needed in the cell, the OM. Cellular machinery has evolved to assist this process; in particular, chaperone proteins in the periplasm called Skp and SurA bind to the newly made OMPs and escort them to the OM. The OMPs are then inserted into the OM and folded to the correct structure by a molecular machine called BAM (beta-barrel assembly machinery). Perturbing this pathway leads to a loss of bacterial viability, and therefore represents a potential new avenue for controlling gram-negative pathogens responsible for infections.
The structures of the chaperone proteins Skp and SurA, and all the component proteins of BAM, are known. What remains undetermined is how these proteins all work together to make new, folded and functional OMPs. We propose to address this fundamental question by exploiting recent exciting developments in mass spectrometry (MS) (along with other biophysical techniques). Our work will focus on how the two chaperones Skp and SurA bind and recognise their substrate OMPs. It is known that Skp contains a large cavity important for trapping OMPs; however, how this cavity is able to accommodate OMPs of different sizes remains unknown. How SurA, a key chaperone, binds and delivers OMPs for folding into the OM is also unknown in molecular detail. Here we will use MS methods to understand how these chaperones function. We will also use similar approaches to determine the structural organisation of BAM for the first time. Finally, in challenging but exciting experiments, we will investigate how chaperone-bound OMPs are delivered to BAM, and how the subunits of BAM rearrange so that OMPs can be inserted into the OM. Finally, we aim to understand how BAM function can be inhibited. The insights into this essential cellular machinery gained from this work may lead to the identification of much-needed new targets for antibiotics against gram-negative bacteria that cause disease to humans, plants and animals.

The beta-barrel outer membrane proteins (OMPs) of gram-negative bacteria are essential for cell viability and survival, but their biogenesis is poorly understood. OMPs are synthesised in the cytoplasm, translocated across the inner membrane and transported across the periplasm by the molecular chaperones Skp or SurA, before being inserted into the OM by the essential BAM complex (a 203 kDa heteropentameric membrane protein complex). The mechanisms by which Skp and SurA recognise OMPs and successfully deliver their cargos to BAM remain poorly understood. These questions are especially interesting since the periplasm is devoid of ATP, and it is completely unknown how substrate binding and release are controlled and coordinated. Moreover, there is currently no structure of the BAM complex, nor is there any detailed knowledge about how OMPs are delivered to BAM from Skp/SurA.
Here we will exploit recent developments in our laboratory in which we have used non-covalent mass spectrometric (MS) methods to show that (i) the stoichiometry of Skp:OMP assemblies varies with OMP size; (ii) the intact BAM complex can be analysed using non-covalent MS and (iii) SurA can be visualised binding to BAM using ESI-MS. Using innovative footprinting and chemical crosslinking methods coupled to MS, combined with native MS and other biophysical techniques, we propose here to address and answer the following questions:

(i) How do Skp/SurA recognise and bind their wide repertoire of OMP substrates?
(ii) What is the structure of the intact BAM complex?
(iii) How does BAM interact with Skp/SurA to take delivery of its OMP substrates before successfully folding them into the OM?

The insights gained will not only inform about a fascinating macromolecular machine that is able to function without an external energy source, but in the long term, may pave the way to develop new strategies to control gram-negative organisms by targeting this essential cellular pathway.

This project addresses the key issue of understanding the complex biochemical pathway that underlies outer membrane protein (OMP) biogenesis in gram-negative bacteria. How this assembly line is achieved is poorly understood, including the mechanism(s) by which the OMP chaperones Skp and SurA function, how unfolded OMPs are recognised by the BAM complex and how BAM itself folds and inserts OMPs into the outer membrane (OM). Especially fascinating is the fact that this protein assembly line functions in the absence of an external energy source (the periplasm is devoid of ATP). The immediate impact of this research will be for researchers interested in membrane proteins, OMP biogenesis and the structure/function relationship of chaperones and macromolecular assemblies (see Academic Beneficiaries).

Equally important is the potential development of our fundamental research to develop much-needed new strategies to combat gram-negative pathogens. Gram-negative bacteria pose major threats to the health and wealth of the UK as they are major pathogens in humans (E. coli, K. pneumoniae, P. aeruginosa, A. baumannii, and Enterobacter sp), livestock (E. coli, D. nodosus, C. abortus and L. intracellularis) and plants (P. syringae, Xanthomonas sp. and E. amylovora). The cost of these infections and nosocomial infections with antibiotic resistant Gram-negative bacteria is estimated to exceed 1.5 billion Euro in healthcare expenses and lost productivity each year in the EU (UK Five Year Antimicrobial Resistance Strategy 2013 to 2018, Depts of Health and Environment, Food and Rural Affairs, Sept 2013). The OMP biogenesis pathway represents a particularly attractive, but currently under-explored route for controlling gram-negative bacteria, since (i) OMP biogenesis is essential for cell viability; (ii) Skp/SurA/BAM are either not present or are not conserved in man; (iii) small molecules (less than 600 Da) can readily diffuse through the OM, and (iv) BamA and BamD (components of BAM) are highly conserved amongst gram-negative bacteria.

Given the urgent unmet need for new antibacterial therapeutics, we plan to exploit every opportunity for interacting with relevant industries and other interested parties as soon as possible during the proposed programme of study so that we can share results and exploit their potentials (see Pathway to Impact). Traditional routes of dissemination will be used for this purpose, including presentations at research conferences, publications, press releases and the web sites of the University, ACSMB and the applicant's research groups. We will also use also every opportunity to communicate our excitement about this research to our contacts in industry (including AZ, GSK, UCB, Medimmune, the latter two with which we have established on-going links) so that potential translational opportunities can be pursued from the initial stages of the project. During the project we will also host in an "Open Day for Industry" which will focus on anti-bacterial strategies, and form part of our established Astbury-Industry activities, where we will further highlight the research and opportunities for exploiting our findings in the development of potential therapies.