الوصف: |
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. Open Access |