Association/Dissociation of the Nucleotide-binding Domains of the ATP-binding Cassette Protein MsbA Measured during Continuous Hydrolysis

Abstract

Background:In ATP-binding cassette proteins, ATP binding produces association of the two nucleotide-binding domains (NBDs), but the molecular mechanism is unknown. Results: The NBDs separate following ATP hydrolysis. Conclusion: NBD dimers dissociate during the hydrolysis cycle supporting monomer/dimer models of operation. Significance: Knowledge of the molecular mechanism of hydrolysis will help us understand how ATP-binding cassette proteins work.

The Lipid Bilayer Modulates the Structure and Function of an ATP-binding Cassette Exporter

“…Several studies point to large motions between the nucleotide-free "open" inward-facing conformation (NBDs separated by tens of Angstroms) and the ATP-bound "closed" outward-facing conformation (tightly associated NBDs). These observations disagree with evidence suggesting that the NBDs remain in close contact at all times during the transport cycle and that such large conformational changes might not be physiological (19 -22).For simplicity and because of technical limitations, essentially all structural studies of ABC exporters have been performed with the proteins solubilized in detergent, locked in specific conformations and/or at low temperature (11,12,14,18,(23)(24)(25)(26). However, understanding of the structure-function of membrane proteins requires their study under more physiological conditions, at a minimum: 1) reconstitution into lipid bilayers, their native environment; 2) physiological temperatures; and 3) functioning conditions, as opposed to specific locked conformations.…”

“…We found that MsbA reconstituted in nanodiscs, and at physiological temperature, showed 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 (11,13,14,18).…”

“…The Glu-506 to Gln mutation was introduced into T561C to generate the catalytically inactive E506Q/T561C mutant. MsbA was expressed and purified essentially as described (18). Briefly, MsbA expression in BL21 DE3-RILP Escherichia coli cells (Agilent Technologies, Santa Clara, CA) was induced with 1 mM isopropyl-␤-D-thiogalactopyranoside for 4 h at 30°C.…”

“…For simplicity and because of technical limitations, essentially all structural studies of ABC exporters have been performed with the proteins solubilized in detergent, locked in specific conformations and/or at low temperature (11,12,14,18,(23)(24)(25)(26). However, understanding of the structure-function of membrane proteins requires their study under more physiological conditions, at a minimum: 1) reconstitution into lipid bilayers, their native environment; 2) physiological temperatures; and 3) functioning conditions, as opposed to specific locked conformations.…”

“…MsbA is located in the inner membrane of Gram-negative bacteria, where it transports lipid A from the inner to the outer leaflet (9,10). MsbA has been crystallized in inward-and outward-facing conformations (11) and has also been extensively studied by different spectroscopic techniques (12)(13)(14)(15)(16)(17)(18). Several studies point to large motions between the nucleotide-free "open" inward-facing conformation (NBDs separated by tens of Angstroms) and the ATP-bound "closed" outward-facing conformation (tightly associated NBDs).…”