Structural insights into the catalytic cycle of a bacterial multidrug ABC efflux pump

Waqas Javed (1, 2, 3) , Sylvain Vallet (2) , Marie-Pierre Clement (2) , Aline Le Roy (1) , Martine Moulin (3) , Michael Härtlein (3) , Cécile Breyton (1) , Odile Burlet-Schiltz (4) , Julien Marcoux (4) , Cédric Orelle (2) , Christine Ebel (1) , Anne Martel (3) , Jean-Michel Jault (2)

1 IBS - UMR 5075 - Institut de biologie structurale
2 MMSB - Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry
3 ILL - Institut Laue-Langevin
4 IPBS - Institut de pharmacologie et de biologie structurale

ABC ("ATP-Binding Cassette") transporters of the type IV subfamily consist of exporters involved in the efflux of many compounds, notably those capable to confer multidrug resistance like the mammalian P-glycoprotein or the bacterial transporter BmrA. They function according to an alternating access mechanism between inward-facing (IF) and outward-facing (OF) conformations, but the extent of physical separation between the two nucleotide-binding domains (NBDs) in different states is still unsettled. Small Angle Neutron Scattering and hydrogen/deuterium exchange coupled to mass spectrometry were used to highlight different conformational states of BmrA during its ATPase cycle. In particular, mutation of the conserved Lysine residue of the Walker-A motif (K380A) captures BmrA in an ATP-bound IF conformation prior to NBD closure. While in the transition-like state induced by vanadate wild-type BmrA is mainly in an OF conformation, the transporter populates only IF conformations in either the apo state or in the presence of ADP/Mg. Importantly, in this post-hydrolytic step, distances between the two NBDs of BmrA seem to be more separated than in the apo state, but they remain shorter than the widest opening found in the related MsbA transporter. Overall, our results highlight the main steps of the catalytic cycle of a homodimeric bacterial multidrug transporter and underline structural and functional commonalities as well as oddities among the type IV subfamily of ABC transporters.

Mots clés: ATP-binding cassette; hydrogen deuterium exchange; membrane protein; multidrug transporter; small angle neutron scattering

Domaines: Biologie structurale [q-bio.BM]

Waqas Javed, Sylvain Vallet, Marie-Pierre Clement, Aline Le Roy, Martine Moulin, et al.. Structural insights into the catalytic cycle of a bacterial multidrug ABC efflux pump. Journal of Molecular Biology, 2022, 434 (9), pp.167541. ⟨10.1016/j.jmb.2022.167541⟩. ⟨hal-03715136⟩