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 ATPbinding 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 dem-

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

“…Colored balls show the position of the cysteines used for probing conformational changes. PBD codes for McjD in inward‐occluded conformation are 5OFP (left; Bountra et al , 2017) and in the outward‐occluded conformation are 4PL0 (right; Choudhury et al , 2014). …”

“…The FRET efficiency changes are related to the distance of the respective variants and thus indicate the changes that occur upon addition of ATP or the substrate MccJ25. For example, the crystal structure estimates that the nucleotide‐free form of McjD should show a larger NBD separation (low FRET), while this should be shorter in the nucleotide‐bound form (high FRET; Choudhury et al , 2014; Bountra et al , 2017). …”

“…Opening of the TMD of heterodimeric ABC transporters is coupled to ATP hydrolysis rather than binding due to the nature of their NBDs that contain a consensus and degenerate site for ATP hydrolysis (Mishra et al , 2014; Timachi et al , 2017; Barth et al , 2018). 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).…”

“…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.…”

“…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?…”