Dissection of the conformational cycle of the multidrug/lipidA ABC exporter MsbA.
Recent crystal structures of the multidrug ATP-binding cassette (ABC) exporters Sav1866 from Staphylococcus aureus, MsbA from Escherichia coli, Vibrio cholera, and Salmonella typhimurium, and mouse ABCB1a suggest a common alternating access mechanism for export. However, the molecular framework underlying this mechanism is critically dependent on assumed conformational relationships between nonidentical crystal structures and therefore requires biochemical verification. The structures of homodimeric MsbA reveal a pair of glutamate residues (E208 and E208') in the intracellular domains of its two half-transporters, close to the nucleotide-binding domains (NBDs), which are in close proximity of each other in the outward-facing state but not in the inward-facing state. Using intermolecular cysteine crosslinking between E208C and E208C' in E. coli MsbA, we demonstrate that the NBDs dissociate in nucleotide-free conditions and come close on ATP binding and ADP·vanadate trapping. Interestingly, ADP alone separates the half-transporters like a nucleotide-free state, presumably for the following catalytic cycle. Our data fill persistent gaps in current studies on the conformational dynamics of a variety of ABC exporters. Based on a single biochemical method, the findings describe a conformational cycle for a single ABC exporter at major checkpoints of the ATPase reaction under experimental conditions, where the exporter is transport active.
Doshi
Rupak
R
Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.
Woebking
Barbara
B
van Veen
Hendrik W
HW
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; C079726:MsbA protein, Bacteria; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D003545:Cysteine