title: Association/dissociation of the nucleotide-binding domains of the ATP-binding cassette protein MsbA measured during continuous hydrolysis.
abstract: In ATP-binding cassette proteins, the two nucleotide-binding domains (NBDs) work as dimers to bind and hydrolyze ATP, but the molecular mechanism of nucleotide hydrolysis is controversial. It is still unresolved whether hydrolysis leads to dissociation of the ATP-induced dimers or partial opening of the dimers such that the NBDs remain in contact during the hydrolysis cycle. We studied the bacterial lipid flippase MsbA by luminescence resonance energy transfer (LRET). The LRET signal between optical probes reacted with single-cysteine mutants was employed to follow NBD association/dissociation in real time. The intermonomer distances calculated from LRET data indicate that the NBDs separate completely following ATP hydrolysis, even in the presence of mm MgATP, and that the dissociation occurs following each hydrolysis cycle. The results support association/dissociation, as opposed to constant contact models, for the mode of operation of ATP-binding cassette proteins.
authors.affiliation: From the Department of Cell Physiology and Molecular Biophysics, and Center for Membrane Protein Research, Texas Tech Health Sciences Center, Lubbock, Texas 79430-6551.
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Cooper
authors.affiliation: From the Department of Cell Physiology and Molecular Biophysics, and Center for Membrane Protein Research, Texas Tech Health Sciences Center, Lubbock, Texas 79430-6551. Electronic address: g.altenberg@ttuhsc.edu.
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Altenberg
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D055162:Biocatalysis; D001896:Boron Compounds / Q000378:metabolism; D004735:Energy Transfer; D006868:Hydrolysis; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D049449:Luminescence; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012486:Salmonella typhimurium / Q000378:metabolism*; D013997:Time Factors
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D001896:Boron Compounds; D008081:Liposomes; C079726:MsbA protein, Bacteria; D050505:Mutant Proteins; D009711:Nucleotides; D000251:Adenosine Triphosphatases
keywords: ABC Transporter; ATPases; CFTR; Dimerization; Fluorescence; Kinetics; LRET; Luminescence Resonance Energy Transfer; Multidrug Transporters