The Lipid Bilayer Modulates the Structure and Function of an Atp-Binding Cassette Exporter

Abstract

ATP-binding cassette (ABC) exporters use the energy of ATP hydrolysis to transport substrates across membranes. ABC proteins contain two transmembrane domains that form the translocation pathway, and two conserved nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. ATP binding promotes NBD dimerization, which is essential for ATP hydrolysis, whereas NBDs dissociation occurs following hydrolysis. This NBD dimerization/dissociation process is coupled to rearrangements of the transmembrane helices, switching the transporters from an inward-facing conformation (dissociated NBDs) to an outward-facing conformation (dimeric NBDs), with the concomitant translocation of substrate. Structural studies of these proteins have been performed with the proteins in detergent micelles, locked in specific conformations and/or at low temperature. As part of our efforts to study ABC proteins under more natural experimental conditions, we used luminescence resonance energy transfer (LRET) on the prototypical ABC exporter MsbA reconstituted in nanodiscs, at 37oC, and while it performs ATP hydrolysis. We have found that, in these native-like conditions, MsbA adopts two main conformations: NBDs in closed proximity (36-Å distance) and NBDs partially separated (∼46-Å distance), with changes in the percentage of molecules adopting each conformation during the ATP hydrolysis cycle. Essentially, our results show major differences with the published crystal structure in the open inward-facing conformation, as well as with double electron-electron resonance (DEER) experiments and LRET experiments performed with MsbA in detergent micelles. The most striking differences include a significantly smaller separation between the NBDs, and a larger fraction of molecules with associated NBDs in the apo state. These studies stress the importance of studying membrane proteins under native-like conditions that include the insertion into lipid bilayers and normal temperatures.