title: Locking TolC entrance helices to prevent protein translocation by the bacterial type I export apparatus.
abstract: The periplasmic entrance of the TolC channel tunnel is sealed by close-packing of inner and outer coiled-coils, and it has been proposed that opening of the entrance is achieved by an iris-like realignment of the inner coiled-coils. This is supported by experimental disruption of the key links connecting them, which effects transition to the open state in TolC inserted into planar lipid bilayers. Here we provide in vivo evidence for this "twist to open" mechanism by constraining the coiled coils with disulphide bonds, either self-locking or bridged by a chemical cross-linker, and reconstituting the resulting TolC variants into the type I protein export system in Escherichia coli. Introducing an intermonomer disulphide bridge between Ala159 and Ser350 caused a fivefold reduction in export, and when the coiled coils were cross-linked at the entrance constriction, between Asp374 of adjacent monomers or between Asn156 and Ala375, TolC-dependent export was abolished. In vivo cross-linking showed that the locked non-exporting TolC variants were still recruited to assemble the type I export apparatus. The data show that untwisting the entrance helices is essential for the export function of TolC in E.coli, specifically to allow access and passage of substrates engaged at the inner membrane translocase.
authors.affiliation: Department of Pathology, Cambridge University, Tennis Court Road, UK.
authors.forename: Jeyanthy
authors.initials: J
authors.lastname: Eswaran
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 / Q000235:genetics / Q000502:physiology*; D018407:Bacterial Physiological Phenomena; D001692:Biological Transport; D004594:Electrophysiology; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; D008954:Models, Biological; D008958:Models, Molecular; D009068:Movement; D019897:Periplasm; D011487:Protein Conformation; D021381:Protein Transport; D012399:Rotation
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli