Professor Colin Hughes
Membrane machineries for toxin export and multidrug efflux
TolC-dependent membrane machineries export large virulence proteins like toxins, or eject antibiotics and other inhibitory agents, so are important to the survival of pathogens like E.coli, Pseudomonas, Neisseria, Serratia and Bordatella. These toxin export systems and multidrug resistance ‘pumps’ all have a tripartite structure, comprising an outer membrane-anchored TolC protein that projects across the inter-membrane periplasmic space to present an exit duct or 'trash chute', to molecules bound by inner membrane transporters. These are typically a traffic ATPase for protein export, and either an ATPase or a proton antiporter for efflux. The two apposed inner and outer membrane components are structurally and functionally linked by a periplasmic adaptor protein that is key to the recruitment and opening of the periplasmic entrance to the TolC exit channel. The assembled pumps span the entire bacterial cell envelope of both membranes and periplasmic space.
We have defined the toxin export signal and identified translocation stages and intermediates of the toxin export mechanism, and shown that when the transporter-adaptor complex is bound by export substrate it recruits TolC to effect reversible assembly. To establish the key features of the export and efflux mechanisms, we elucidated by crystallography high resolution structures of the common proteins, the TolC exit duct and the efflux pump adaptor. By extensive in vivo site-specific cross-linking and multidomain docking we established the interaction interfaces between the efflux pump exit duct, adaptor and antiporter. This has allowed us to use complex modelling to present the first view of a c.600,000 Da assembled multidrug efflux machinery. By combining electrophysiology of purified TolC open state variants inserted in lipid bilayers with the crystallography of closed and sequential open states we are elucidating the coiled coil movements underlying the ‘iris-like’ mechanism TolC entrance opening mechanism, and how this could be triggered and stabilized by the periplasmic hairpin of the efflux pump adaptor. Similar approaches have shown the TolC entrance can be liganded and blocked, indicating a potential way for pumps to be inhibited by novel drugs. This work is codirected by Prof. Koronakis.
The flagella subunit export pathway underlying assembly
Motile pathogenic bacteria build multi-component rotating flagella 'nanomachines' that act like helical 'propellers' on their surface to effect cell movement and population migration. In bacteria such as Salmonella, the flagellar filament comprises about 20,000 subunits, which pass through the central channel within the growing structure and polymerize under the flagella cap, following assembly of the flexible hook structure. Our research addresses the ordered process by which the flagellar structural subunits, those for the filament, cap, and filament-hook junction, are exported from the cytosol and delivered to the growing flagellar on the cell surface. This is achieved by a complex membrane export apparatus comprising over a dozen different proteins, including those of the integral inner membrane structure, the ATPase complex, and three chaperones that bind the major filament subunits, or minor cap or filament junction subunits. By assembling in vitro complexes and using genetics to create stalled intermediate export complexes we revealed how chaperones protect and pilot flagellar structural subunits to dock at an oligomeric membrane export ATPase. We have revealed an unusual mechanism in which the chaperones of the minor subunits are then selectively cycled at the ATPase complex by an escort protein, a process we suggest enhances subunit export and also facilitates the ordered stoichiometric assembly of the flagellum. This work is codirected by Dr Fraser.
Work is funded by parallel Programme Grants from the Wellcome Trust.