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HDX-MS studies of the bacterial multidrug ABC transporter BmrA in presence or absence of a ligand (rhodamine-6G)

ABSTRACT: Multidrug ATP-Binding Cassette (ABC) exporters use an alternating access mechanism to translocate drugs across membranes, but the molecular mechanism of substrate release is still elusive due to the poor affinity of the drug-binding pocket for its ligand at this stage. Here, we bring new information on this mechanism by resolving two ATP-bound outward-facing conformations of the homodimer BmrA from Bacillus subtilis by X-ray crystallography with no drug bound and by cryo-EM in the presence of rhodamine 6G. This revealed the first structure of a multidrug pump with a substrate bound to the drug-binding pocket in a pre-release state. Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5’-6’ of the other. Hydrogen-Deuterium exchange coupled to mass spectrometry (HDX-MS) and molecular dynamics simulations confirmed the high flexibility of this region, presumably driving the release of structurally divergent compounds.

INSTRUMENT(S): Synapt MS

ORGANISM(S): Bacillus Subtilis

SUBMITTER: Julien Marcoux

LAB HEAD: Julien Marcoux

PROVIDER: PXD027447 | Pride | 2022-01-27

REPOSITORIES: Pride

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