Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state

DOI: 10.1073/pnas.1320506111
PMID: 24920594

Authors: Hassan Choudhury (Imperial College London) , Zhen Tong (University of Cambridge) , Indran Mathavan (Imperial College London) , Yanyan Li (Centre National de la Recherche Scientifique) , So Iwata (Diamond Light Source) , Séverine Zirah (Centre National de la Recherche Scientifique) , Sylvie Rebuffat (Centre National de la Recherche Scientifique) , Hendrik W. Van Veen (University of Cambridge) , Konstantinos Beis (Diamond Light Source)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences , VOL 111 (25) , PAGES 9145 - 9150

State: Published (Approved)
Published: June 2014
Diamond Proposal Number(s): 6384

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.

Journal Keywords: Amino; Bacteriocins ; Crystallography ; X-Ray ; Escherichia; Mutagenesis ; Site-Directed ; Mutation ; Missense ; Protein; Quaternary ; Protein; Secondary ; Protein; Tertiary

Subject Areas: Biology and Bio-materials


Instruments: I02-Macromolecular Crystallography

Added On: 16/09/2014 09:23

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