HDX-MS describes different catalytic conformations of an ABC transporter involved in multidrug resistance

The emergence of antibiotic resistant “superbugs” is a real threat for our society and ATP Binding Cassette (ABC) transporters is one of the family of integral membrane proteins that can expel cytotoxic drugs from bacteria, thereby reducing their efficacy. ABC transporters such as BmrA from Bacillus subtilis alternate between two conformations in an ATP-dependent manner: the inward-facing (IF) conformation that allows the binding of the drug and ATP, and the outward-facing (OF) conformation, that is generated upon ATP binding and allows the release of the drug outside the cell. Then, ATP hydrolysis restores the IF conformation.

Despite this overall understanding of the catalytic cycle, and the numerous X-ray and cryo-EM structures of ABC transporters in both IF and OF conformations, the degree of opening of the Nucleotide Binding Domains (NBDs), the coupling between the NBDs and TransMembrane (TMs) domains and the degree of flexibility of the TMs regarding drug release are still poorly understood. To address these questions, we performed HDX-MS on WT BmrA and on a series of different catalytically inactive mutants, in the absence or presence of ATP and/or drugs and in detergent micelles and lipid nanodiscs. This work was conducted in collaboration with the group of Jean-Michel Jault and Cédric Orelle (MMSB, IBCP, Lyon) (Chaptal et al. 2022; Javed et al. 2022).

Chaptal V*, Zampieri V*, Wiseman B*, Orelle C, Martin J, Nguyen KA, Gobet A, Di Cesare M, Magnard S, Javed W, Eid J, Kilburg A, Peuchmaur M, Marcoux J, Monticelli L, Högbom M, Schoehn G, Jault JM, Boumendjel A, Falson P* (2022) “Substrate-bound and substrate-free outward-facing structures of a multidrug ABC exporter” Science Advances 8(4):eabg9215.

Javed W, Vallet S, Clement MP, Le Roy A, Moulin M, Härtlein M, Breyton C, Burlet-Schiltz O, Marcoux J*, Orelle C*, Ebel C*, Martel* A, Jault JM* (2022) “Structural insights into the catalytic cycle of a bacterial multidrug ABC efflux pump” Journal of Molecular Biology 167541.