Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg ²⁺ –bound outward-facing conformations of the Bacillus subtilis (homodimeric) BmrA by x-ray crystallography and single-particle cryo–electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in B. subtilis . 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. They induce a rearrangement of TM1–2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.

Overexpression of correctly folded membrane proteins is a fundamental prerequisite for functional and structural studies. One of the most commonly used expression systems for the production of membrane proteins is Escherichia coli. While misfolded proteins typically aggregate and form inclusions bodies, membrane proteins that are addressed to the membrane and extractable by detergents are generally assumed to be properly folded. Accordingly, GFP fusion strategy is often used as a fluorescent proxy to monitor their expression and folding quality. Here we investigated the functionality of two different multidrug ABC transporters, the homodimer BmrA from Bacillus subtilis and the heterodimer PatA/PatB from Streptococcus pneumoniae, when produced in several E. coli strains with T7 expression system. Strikingly, while strong expression in the membrane of several strains could be achieved, we observed drastic differences in the functionality of these proteins. Moreover, we observed a general trend in which mild detergents mainly extract the population of active transporters, whereas a harsher detergent like Fos-choline 12 could solubilize transporters irrespective of their functionality. Our results suggest that the amount of T7 RNA polymerase transcripts may indirectly but notably impact the structure and activity of overexpressed membrane proteins, and advise caution when using GFP fusion strategy.

The detailed mechanism of ATP hydrolysis in ATP-binding cassette (ABC) transporters is still not fully understood. Here, we employed ³¹P solid-state NMR to probe the conformational changes and dynamics during the catalytic cycle by locking the multidrug ABC transporter BmrA in prehydrolytic, transition, and posthydrolytic states, using a combination of mutants and ATP analogues. The ³¹P spectra reveal that ATP binds strongly in the prehydrolytic state to both ATP-binding sites as inferred from the analysis of the nonhydrolytic E504A mutant. In the transition state of wild-type BmrA, the symmetry of the dimer is broken and only a single site is tightly bound to ADP:Mg²⁺:vanadate, while the second site is more 'open' allowing exchange with the nucleotides in the solvent. In the posthydrolytic state, weak binding, as characterized by chemical exchange with free ADP and by asymmetric ³¹P-³¹P two-dimensional (2D) correlation spectra, is observed for both sites. Revisiting the ¹³C spectra in light of these findings confirms the conformational nonequivalence of the two nucleotide-binding sites in the transition state. Our results show that following ATP binding, the symmetry of the ATP-binding sites of BmrA is lost in the ATP-hydrolysis step, but is then recovered in the posthydrolytic ADP-bound state.