Bidirectional signaling between nucleotide and substrate binding sites in a type IV multidrug ABC transporter

ATP-binding cassette (ABC) transporters use ATP to transport substrates across cellular membranes. In type IV ABC transporters, which includes many multidrug resistance (MDR) pumps, communication between the nucleotide-binding domains (NBDs) and transmembrane domains (TMDs) occurs via intracellular coupling helices. However, the precise mechanism of interdomain crosstalk and coordination between ATP and substrate binding sites remain unclear. Here, we combined NMR spectroscopy, Hydrogen-Deuterium eXchange Mass Spectrometry (HDX-MS), PET-FCS and functional assays to identify a conserved residue cluster at the NBD/TMD interface of the bacterial MDR transporter BmrA. This cluster functions as a bidirectional relay to convey nucleotide and substrate occupancy between the two domains through coupling helix 2. Mutations impact both local and global transporter dynamics. Our findings reveal a novel interdomain communication pathway in type IV ABC transporters, offering insights into the coordination of ligand binding in this important protein family.