title: Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state.
abstract: Enterobacteriaceae produce antimicrobial peptides for survival under nutrient starvation. Microcin J25 (MccJ25) is an antimicrobial peptide with a unique lasso topology. It is secreted by the ATP-binding cassette (ABC) exporter McjD, which ensures self-immunity of the producing strain through efficient export of the toxic mature peptide from the cell. Here we have determined the crystal structure of McjD from Escherichia coli at 2.7-Å resolution, which is to the authors' knowledge the first structure of an antibacterial peptide ABC transporter. Our functional and biochemical analyses demonstrate McjD-dependent immunity to MccJ25 through efflux of the peptide. McjD can directly bind MccJ25 and displays a basal ATPase activity that is stimulated by MccJ25 in both detergent solution and proteoliposomes. McjD adopts a new conformation, termed nucleotide-bound outward occluded. The new conformation defines a clear cavity; mutagenesis and ligand binding studies of the cavity have identified Phe86, Asn134, and Asn302 as important for recognition of MccJ25. Comparisons with the inward-open MsbA and outward-open Sav1866 structures show that McjD has structural similarities with both states without the intertwining of transmembrane (TM) helices. The occluded state is formed by rotation of TMs 1 and 2 toward the equivalent TMs of the opposite monomer, unlike Sav1866 where they intertwine with TMs 3-6 of the opposite monomer. Cysteine cross-linking studies on the McjD dimer in inside-out membrane vesicles of E. coli confirmed the presence of the occluded state. We therefore propose that the outward-occluded state represents a transition intermediate between the outward-open and inward-open conformation of ABC exporters.
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom;Membrane Protein Lab, Diamond Light Source, Harwell Science and Innovation Campus, Chilton OX11 0DE, United Kingdom;Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0FA, United Kingdom;
authors.forename: Hassanul G
authors.initials: HG
authors.lastname: Choudhury
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom;
authors.forename: Zhen
authors.initials: Z
authors.lastname: Tong
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom;Membrane Protein Lab, Diamond Light Source, Harwell Science and Innovation Campus, Chilton OX11 0DE, United Kingdom;Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0FA, United Kingdom;
authors.forename: Indran
authors.initials: I
authors.lastname: Mathavan
authors.affiliation: Laboratory of Communication Molecules and Adaptation of Microorganisms, Unité Mixte de Recherche 7245, Muséum National d'Histoire Naturelle, Centre National de la Recherche Scientifique, 75005 Paris, France; and.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Li
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom;Membrane Protein Lab, Diamond Light Source, Harwell Science and Innovation Campus, Chilton OX11 0DE, United Kingdom;Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0FA, United Kingdom;Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.affiliation: Laboratory of Communication Molecules and Adaptation of Microorganisms, Unité Mixte de Recherche 7245, Muséum National d'Histoire Naturelle, Centre National de la Recherche Scientifique, 75005 Paris, France; and.
authors.forename: Séverine
authors.initials: S
authors.lastname: Zirah
authors.affiliation: Laboratory of Communication Molecules and Adaptation of Microorganisms, Unité Mixte de Recherche 7245, Muséum National d'Histoire Naturelle, Centre National de la Recherche Scientifique, 75005 Paris, France; and kbeis@imperial.ac.uk hwv20@cam.ac.uk rebuffat@mnhn.fr.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Rebuffat
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom; kbeis@imperial.ac.uk hwv20@cam.ac.uk rebuffat@mnhn.fr.
authors.forename: Hendrik W
authors.initials: HW
authors.lastname: van Veen
authors.affiliation: Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, United Kingdom;Membrane Protein Lab, Diamond Light Source, Harwell Science and Innovation Campus, Chilton OX11 0DE, United Kingdom;Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot OX11 0FA, United Kingdom; kbeis@imperial.ac.uk hwv20@cam.ac.uk rebuffat@mnhn.fr.
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Beis
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D001430:Bacteriocins; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D018528:ATP-Binding Cassette Transporters; D001430:Bacteriocins; D029968:Escherichia coli Proteins; C020411:microcin
keywords: antimicrobial peptide ABC exporter; membrane transporter crystal structure; microcin immunity protein; substrate binding; transport mechanism