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.

2Laurylmaltose neopentylglycol (LMNG) bears two linked hydrophobic chains of equal length 3 and two hydrophilic maltoside groups. It arouses a strong interest in the field of membrane 4 protein biochemistry, since it was shown to efficiently solubilize and stabilize membrane 5 proteins often better than the commonly used dodecylmaltopyranoside (DDM), and to allow 6 structure determination of some challenging membrane proteins. However, LMNG was 7 described to form large micelles, which could be unfavorable for structural purposes. We thus 8 investigated its auto-assemblies and the association state of different membrane proteins 9 solubilized in LMNG by analytical ultracentrifugation, size exclusion chromatography 10 coupled to light scattering, centrifugation on sucrose gradient and/or small angle scattering. 11 At high concentrations (in the mM range) LMNG forms long rods, and it stabilized the 12 membrane proteins investigated herein, i.e. a bacterial multidrug transporter, BmrA; a 13 prokaryotic analogous of the eukaryotic NADPH oxidases, SpNOX; an E. coli outer 14 membrane transporter, FhuA; and the halobacterial bacteriorhodopsin, bR. BmrA, in the Apo 15 and the vanadate-inhibited forms showed reduced kinetics of limited proteolysis in LMNG 16 compared to DDM. Both SpNOX and BmrA display an increased specific activity in LMNG 17 compared to DDM. The four proteins form LMNG complexes with their usual quaternary 18 structure and with usual amount of bound detergent. No heterogeneous complexes related to 19 the large micelle size of LMNG alone were observed. In conditions where LMNG form 20 assemblies of large size, FhuA crystals diffracting to 4.0 Å were obtained by vapor diffusion. 21 LMNG large micelle size thus does not preclude membrane protein homogeneity and 22 crystallization.23 24 29Detergents are essential for membrane protein solubilization and purification steps, as well as 30 functional and structural studies. However, they often lead to membrane protein inactivation. 31One of the mechanisms leading to inactivation (for a recent review, see [2]) is the dissociation 32 of subunits, loss of essential lipids or other hydrophobic co-factors. As an example, 33 cytochrome b 6 f inactivation and monomerization was related to lipid loss [3]. Detergent 34 dynamics, i.e. detergent exchange between the free monomers, or between micelles, and 35 bound detergent at the surface of the transmembrane protein surface, could trigger transient 36 exposure of this hydrophobic surface leading to irreversible protein aggregation. Detergent 37 binding can also alter protein structure: molecular dynamics simulation indeed showed, for a 38 thermostable mutant of the adenosine receptor, that the harsh octylglucoside detergent 39 molecules intercalate between trans-membrane helices, moving them apart in the 200 ns 40 simulation [4]. The structure and dynamics of different -helical membrane proteins 41 solubilized in alkyl phosphocholines appear deeply altered [5]. For example, NMR, molecular 42 dynamics simulations, and fu...