Resting state conformation of the MsbA homodimer as studied by site-directed spin labeling.
MsbA is the ABC transporter for lipid A and is found in the inner membranes of Gram-negative bacteria such as Escherichia coli. Without MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang, and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, many questions remain concerning its mechanism of transport. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful approach for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions within a protein. The quaternary structure of the resting state of the MsbA homodimer reconstituted into lipid membranes has been evaluated by SDSL EPR spectroscopy and chemical cross-linking techniques. SDSL and cross-linking results are consistent with the controversial resting state conformation of the MsbA homodimer found in the crystal structure, with the tips of the transmembrane helices forming a dimer interface. The position of MsbA in the membrane bilayer along with the relative orientation of the transmembrane helical bundles with respect to one another has been determined. Characterization of the resting state of the MsbA homodimer is essential for future studies on the functional dynamics of this membrane transporter.
Buchaklian
Adam H
AH
Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226, USA.
Funk
Andrea L
AL
Klug
Candice S
CS
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D050356:Lipid Metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D020836:Protein Structure, Quaternary
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; C079726:MsbA protein, Bacteria; D003545:Cysteine