A salt bridge pre-arranges the structure of the ABC transporter BmrA for proper NBD dimerization

• The linker connecting the TMD and NBD of BmrA lies near both coupling helices. • The linker residue Glu326 is essential for the overall BmrA function. • Substitution of Glu326 against Ala results in a disturbed TMD-NBD arrangement. • A conserved salt-bridge between Lys217 and Glu326 pre-arranges the BmrA structure. Substrate translocation across membranes mediated by ATP-binding cassette (ABC) transporters relies on communication between the nucleotide-binding domains (NBDs) and transmembrane domains (TMDs). In type IV ABC exporters, the NBD-TMD communication involves two coupling helices (CHs), yet how this interface is stabilized remains unclear. In Bacillus multidrug resistance ATP (BmrA), the linker connecting the TMD and NBD lies adjacent to both CHs, suggesting a role in maintaining this interface. Via Ala mutagenesis, we analyzed a crucial part of the linker and identified Glu326 being essential, as its substitution impaired the transport activity and abolished the ( in vitro) ATPase activity. Cryo–EM analysis of the Glu326Ala variant revealed a disrupted NBD–TMD arrangement, with the CHs spatially uncoupled from the NBDs. Further mutagenesis pointed to Lys217 in CH2 as a potential interaction partner. We propose that a conserved Glu326-Lys217 salt bridge stabilizes the CH–NBD interface, thereby promoting NBD dimerization and functional coupling. Conservation of this motif across bacterial exporters and human ABC-C family members highlights a critical, previously overlooked role of the linker region.