title: A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps.
abstract: Bacteria such as Escherichia coli and Pseudomonas aeruginosa expel antibiotics and other inhibitors via tripartite multidrug efflux pumps spanning the inner and outer membranes and the intervening periplasmic space. A key event in pump assembly is the recruitment of an outer membrane-anchored TolC exit duct by the adaptor protein of a cognate inner membrane translocase, establishing a contiguous transenvelope efflux pore. We describe the underlying interaction of juxtaposed periplasmic exit duct and adaptor coiled-coils in the widespread RND-type pump TolC/AcrAB of E. coli, using in vivo cross-linking to map the extent of intermolecular contacts. Cross-linking of site-specific TolC cysteine variants to wild-type AcrA adaptor identified residues on the lower alpha-helical barrel domain of TolC, defining a contiguous cluster close to the entrance aperture of the exit duct. Reciprocally, site-specific cross-linking of AcrA cysteine variants to wild-type TolC identified the interaction surface on the adaptor within the N-terminal alpha-helix of the AcrA coiled-coil. The experimental data allowed a data-driven docking approach to model the interaction surface central to pump assembly. The lowest energy docked model satisfying all of the cross-link distance constraints places the adaptor at the intramolecular groove formed by the TolC entrance helices, aligning the adaptor coiled-coil with the exposed TolC outer helix. A key feature of this positioning is that it allows space for the proposed movement of the inner coil of TolC during transition to its open state.
authors.affiliation: Department of Pathology, Cambridge University, Tennis Court Road, Cambridge CB2 1QP, United Kingdom.
authors.forename: Sune
authors.initials: S
authors.lastname: Lobedanz
authors.forename: Evert
authors.initials: E
authors.lastname: Bokma
authors.forename: Martyn F
authors.initials: MF
authors.lastname: Symmons
authors.forename: Eva
authors.initials: E
authors.lastname: Koronakis
authors.forename: Colin
authors.initials: C
authors.lastname: Hughes
authors.forename: Vassilis
authors.initials: V
authors.lastname: Koronakis
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000502:physiology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000502:physiology*; D014644:Genetic Variation; D008074:Lipoproteins / Q000737:chemistry; D008301:Maleimides / Q000494:pharmacology; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D003545:Cysteine; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D008301:Maleimides; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083497:AcrA protein, E coli; C083496:AcrB protein, E coli; C073126:tolC protein, E coli; C043592:maleimide