Under conditions of nutrient starvation bacteria produce and release antibacterial peptides like
the lasso peptide microcin J25 (MccJ25). Uptake of MccJ25 by other bacteria leads to RNA
polymerase inhibition and subsequent cell death. MccJ25 is also toxic to the producing
organism that utilises ATP-binding cassette (ABC) transporters to provide self-immunity. The
ABC transporter McjD is responsible for the efflux of MccJ25. The general architecture of an
ABC transporter comprises two nucleotide-binding domains (NBDs) and two transmembrane
domains (TMDs). Previously, the structure of McjD from Escherichia coli was determined in
complex with a non-hydrolysable ATP analogue (AMP-PNP), providing some insights into the
transport cycle. However, the mechanistic basis for MccJ25 secretion remained elusive. In this
work, the structure of McjD has been determined in a post-ATP hydrolysis intermediate state
(ADP-VO4). Using predictive cysteine cross-linking, cavity accessibility studies, transport
assays and PELDOR measurements in lipid membranes, a novel mechanism for MccJ25
secretion is proposed requiring the transient opening of the McjD TMDs for substrate release.
Unlike multidrug ABC exporters which display large conformational changes in the TMDs,
the McjD TMDs exist in a predominantly occluded state which prevents MccJ25 reuptake upon
efflux. These structural insights are complimented by the first single molecule FRET (smFRET)
characterisation of an ABC exporter in a native-like environment. The smFRET findings report
conformational changes in the NBDs and TMDs of McjD, demonstrating that opening of the
TMDs is tightly coupled to the binding of both ATP and MccJ25. The NBDs display intrinsic
conformational dynamics on the millisecond timescale whereas the TMDs do not show any
dynamic behaviour. Finally, attempts are made to purify and functionally characterize two
staphylococcal peptide ABC exporters Pmt and AbcA. These transporters secrete cytolytic α-
helical peptides, phenol soluble modulins, that can evade the immune response.