title: Outer-membrane translocation of bulky small molecules by passive diffusion.
abstract: The outer membrane (OM) of gram-negative bacteria forms a protective layer around the cell that serves as a permeability barrier to prevent unrestricted access of noxious substances. The permeability barrier of the OM results partly from the limited pore diameters of OM diffusion channels. As a consequence, there is an "OM size-exclusion limit," and the uptake of bulky molecules with molecular masses of more than ∼ 600 Da is thought to be mediated by TonB-dependent, active transporters. Intriguingly, the OM protein CymA from Klebsiella oxytoca does not depend on TonB but nevertheless mediates efficient OM passage of cyclodextrins with diameters of up to ∼ 15 Å. Here we show, by using X-ray crystallography, molecular dynamics simulations, and single-channel electrophysiology, that CymA forms a monomeric 14-stranded β-barrel with a large pore that is occluded on the periplasmic side by the N-terminal 15 residues of the protein. Representing a previously unidentified paradigm in OM transport, CymA mediates the passive diffusion of bulky molecules via an elegant transport mechanism in which a mobile element formed by the N terminus acts as a ligand-expelled gate to preserve the permeability barrier of the OM.
authors.affiliation: Institute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom; bert.van-den-berg@ncl.ac.uk.
authors.forename: Bert
authors.initials: B
authors.lastname: van den Berg
authors.affiliation: School of Engineering and Science, Jacobs University Bremen, 28759 Bremen, Germany.
authors.forename: Satya
authors.initials: S
authors.lastname: Prathyusha Bhamidimarri
authors.affiliation: School of Engineering and Science, Jacobs University Bremen, 28759 Bremen, Germany.
authors.forename: Jigneshkumar
authors.initials: J
authors.lastname: Dahyabhai Prajapati
authors.affiliation: School of Engineering and Science, Jacobs University Bremen, 28759 Bremen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Kleinekathöfer
authors.affiliation: School of Engineering and Science, Jacobs University Bremen, 28759 Bremen, Germany.
authors.forename: Mathias
authors.initials: M
authors.lastname: Winterhalter
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D018360:Crystallography, X-Ray; D003505:Cyclodextrins / Q000378:metabolism; D004058:Diffusion; D041121:Klebsiella oxytoca / Q000378:metabolism*; D056004:Molecular Dynamics Simulation
chemical_list: D001425:Bacterial Outer Membrane Proteins; D003505:Cyclodextrins
keywords: CymA; cyclodextrin; ligand gating; outer membrane channel; passive diffusion