Structural Insights into the Catalytic Cycle of a Bacterial Multidrug ABC Efflux Pump

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 Pglycoprotein 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.(c) 2022 Elsevier Ltd. All rights reserved.

Automated summary

This study used small angle neutron scattering and hydrogen/deuterium exchange coupled to mass spectrometry to investigate the conformational states of the BmrA transporter during its ATPase cycle. The researchers found that under specific mutation conditions, the BmrA transporter can be trapped in an ATP-bound inward-facing conformation. Comparing the transition-like state induced by vanadate with the apo state or the presence of ADP/Mg, it was observed that in the post-hydrolytic step, the distances between the two nucleotide-binding domains appeared to be more separated but remained shorter than in the widest opening state found in the related transporter MsbA.