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

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.Microcins are gene-encoded antibacterial peptides of low molecular weight (<10 kDa), produced by Enterobacteriacea (1). They are secreted under conditions of nutrient exhaustion through dedicated ATP-binding cassette (ABC) exporters and exert potent antibacterial activity against closely related species (2). Microcin J25 (MccJ25) is a plasmid-encoded, ribosomally synthesized, and posttranslationally modified 21-aa antimicrobial peptide (3). Its 3D structure shows a unique lasso topology (4–6), with the C-terminal tail threading through an N-terminal eight-residue macrolactam ring, where it is locked by bulky amino acid side chains, thus forming a compact interlocked structure called the lasso fold (Fig. S1). This extraordinarily stable structure is apportioned into two regions: a loop involved in uptake of the microcin into sensitive bacteria and a ring/tail region that interacts with the cytoplasmic target of the antimicrobial peptide (1, 7, 8). MccJ25 enters the target cell using the siderophore receptor FhuA (9), and inside the cell it inhibits the bacterial RNA polymerase (7, 8, 10). Four genes are required for the biosynthesis and export of MccJ25 (11). The lasso topology is acquired by modification of a linear 58-aa precursor peptide (McjA) by two dedicated enzymes (McjB and McjC) (12). The ABC transporter McjD ensures efficient export of the toxic mature peptide out of the cell and simultaneously serves as a self-immunity strategy for the producing strain (11). Homologs of McjD and MccJ25-like defense systems can be identified in several genomes of bacterial pathogens (13).ABC exporters form a large superfamily of transmembrane proteins responsible for the translocation across the membrane of a large diversity of substrates, ranging from small ions to amino acids, sugars, lipids, or peptides, using the energy of ATP hydrolysis. Some ABC exporters contribute to multidrug resistance. Bacterial ABC exporters are dimers, with each monomer composed of a transmembrane domain (TMD) consisting of six TM helices, which forms the translocation pathway across the membrane bilayer and ensures the substrate specificity, and a nucleotide-binding domain (NBD) where binding and hydrolysis of ATP take place. Biochemical and modeling studies, and the crystal structures of the Escherichia coli lipid A transporter MsbA (14), the Staphylococcus aureus exporter Sav1866 (15), and others suggest that ABC exporters extrude their substrates out of the cell via an alternating access mechanism. However, the current structures do not explain how the transition between inward-open and outward-open conformations occurs mechanistically. Here we have determined the high-resolution structure at 2.7-Å resolution of the E. coli immunity-conferring ABC exporter McjD that is responsible for the export of the lasso peptide MccJ25. It displays a new conformation, outward-occluded and without intertwining of the TMDs, which is intermediate between the outward-open and inward-open state. In addition, the structure defines a clear binding cavity that can accommodate one MccJ25 molecule. Our functional data in detergent solution and proteoliposomes demonstrate that McjD mediates MccJ25 transport in an ATP-dependent fashion and that in the absence of Mccj25, the protein can mediate the transport of typical substrates of multidrug transporters.