Drug-bound and -free outward-facing structures of a multidrug ABC exporter point to a swing mechanism

Abstract

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 two ATP-Mg2+-bound outward-facing (OF) conformations of the Bacillus subtilis (homodimeric) BmrA, one by X-ray crystallography without drug, and another by single-particle cryo-EM with rhodamine 6G (R6G). 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[prime]-6[prime] of the other. R6G induces a rearrangement of TM1-2, highlighting a flexibility that was confirmed by H/D exchange and molecular dynamics simulations. The latter also shows a fast post-release occlusion of the cavity driven by hydrophobicity. Altogether, these data support a new swing mechanism for drug transport.

The Det.Belt Server: A Tool to Visualize and Estimate Amphipathic Solvent Belts around Membrane Proteins

“…Detergents are often used in mixtures, and it is possible to represent several detergents forming the belt, each having their own number of monomers. We must emphasize that detergent mixtures do not behave as single detergents, and also do not behave around the protein as they behave in mixed micelles [ 36 ]. Very few data are available on detergent mixture quantification; an example of the mixture DDM and cholate has been extensively described in [ 36 ] ( Figure 3 ).…”

“…We must emphasize that detergent mixtures do not behave as single detergents, and also do not behave around the protein as they behave in mixed micelles [ 36 ]. Very few data are available on detergent mixture quantification; an example of the mixture DDM and cholate has been extensively described in [ 36 ] ( Figure 3 ). We chose to represent detergent mixtures as stacked cylinders, with each stack representing the volume occupied by the input number of each detergent.…”

“…Similarly, we opted to render detergent mixtures as stacked cylinders showing the portion of the volume occupied by each detergent with the idea to stress caution in the interpretation of the data. Indeed, belts consisting of detergent mixtures have rarely been deeply investigated, and yielded many surprises when thoroughly quantified [ 36 ]. The server is thus not rendering detergent as belts radiating away from the protein, as this can lead to misinterpretation on the positioning of each detergent within the mixture.…”

“…For a more physiological representation of the detergent mixture, investigators are encouraged to explore this behavior using molecular dynamics simulations. [36]. The ratios define the detergent's molar ratio used during the purification.…”

“… Representation of detergent mixtures. ( A ) Purification condition for the ABC transporter BmrA as described in [ 36 ]. The ratios define the detergent’s molar ratio used during the purification.…”