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.

To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl-substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane-cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b, 8 c, and 9 b preserved the ATPase function of BmrA, an ATP-binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a, 8 b, 8 f, 9 a, and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G-protein-coupled adenosine receptor A R. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs.