Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

Abstract

Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.

Conformational dynamics of the ABC transporter McjD seen by single‐molecule FRET

“…In contrary, we have previously shown that the antibacterial peptide ABC transporter McjD, which confers bacterial cells with self‐immunity against the antibacterial peptide MccJ25, adopts distinct occluded conformations (Choudhury et al , 2014; Bountra et al , 2017) to other bacterial exporters including MsbA (Ward et al , 2007). The TMD is occluded to either side of the membrane in the presence or absence of nucleotides that was also supported by Pulsed Electron‐Electron Double Resonance (PELDOR) measurements in bicelles (Bountra et al , 2017). Occluded conformations have also been reported for other ABC transporters (Lin et al , 2015; Perez et al , 2015; Morgan et al , 2017).…”

“…All ABC transporters share a common architecture consisting of a transmembrane domain (TMD) for substrate recognition and transport, and a nucleotide‐binding domain (NBD) that converts the chemical energy of ATP into conformational changes for transport (Beis, 2015). The structures of several homodimeric (Dawson & Locher, 2006; Ward et al , 2007; Perez et al , 2015) and heterodimeric ABC transporters (Hohl et al , 2012; Noll et al , 2017) revealed distinct conformations and suggest, in combination with biophysical studies (e.g., EPR and NMR; Dong et al , 2005; Zou et al , 2009; Bountra et al , 2017; Timachi et al , 2017; Barth et al , 2018), that they undergo large conformational changes during transport. Their complex architecture is, however, a fundamental hurdle to fully understand the coupling between conformational changes, substrate binding, ATP binding and hydrolysis, and transport.…”

“…Based on the available structural, biophysical, and biochemical data for exporters, two transport mechanisms have been proposed, the alternating access mechanism (Dawson & Locher, 2006; Ward et al , 2007; Choudhury et al , 2014; Bountra et al , 2017) and the outward‐only mechanism (Perez et al , 2015). ABC exporters that use the alternating access mechanism switch between inward‐ and outward‐facing states with transmembrane helices intertwining, which exposes the ligand binding site alternatively to the inside or outside of the membrane, a process that is believed to be coupled to ATP binding to hydrolysis.…”

“…Occluded conformations have also been reported for other ABC transporters (Lin et al , 2015; Perez et al , 2015; Morgan et al , 2017). We have biochemically shown that McjD adopts a transient outward‐open conformation that it is probably not long‐lived or well populated to be trapped by PELDOR (Bountra et al , 2017). Although both MsbA and McjD utilize the alternating access mechanism for export of their substrate, the overall mechanism is not fully conserved and requires further characterization.…”

“…In this study, it was used to answer the following mechanistic questions regarding McjD: (i) How is ATP binding or hydrolysis coupled to the opening of the TMD? In McjD, crystal structures and PELDOR data in the presence of the ATP‐analogue adenosine 5′‐(β,γ‐imido)triphosphate (AMPPNP), mimicking ATP binding, and ADP‐vanadate (mimicking ATP hydrolysis) could not reveal an open cavity (Choudhury et al , 2014; Bountra et al , 2017). (ii) What are the intrinsic and ligand‐induced conformational dynamics of McjD as a model system for ABC exporters and on what timescales do they occur?…”