title: Time-resolved Fourier transform infrared spectroscopy of the nucleotide-binding domain from the ATP-binding Cassette transporter MsbA: ATP hydrolysis is the rate-limiting step in the catalytic cycle.
abstract: MsbA is an essential Escherichia coli ATP-binding cassette (ABC) transporter involved in the flipping of lipid A across the cytoplasmic membrane. It is a close homologue of human P-glycoprotein involved in multidrug resistance, and it similarly accepts a variety of small hydrophobic xenobiotics as transport substrates. X-ray structures of three full-length ABC multidrug exporters (including MsbA) have been published recently and reveal large conformational changes during the transport cycle. However, how ATP hydrolysis couples to these conformational changes and finally the transport is still an open question. We employed time-resolved FTIR spectroscopy, a powerful method to elucidate molecular reaction mechanisms of soluble and membrane proteins, to address this question with high spatiotemporal resolution. Here, we monitored the hydrolysis reaction in the nucleotide-binding domain of MsbA at the atomic level. The isolated MsbA nucleotide-binding domain hydrolyzed ATP with V(max) = 45 nmol mg(-1) min(-1), similar to the full-length transporter. A Hill coefficient of 1.49 demonstrates positive cooperativity between the two catalytic sites formed upon dimerization. Global fit analysis of time-resolved FTIR data revealed two apparent rate constants of ~1 and 0.01 s(-1), which were assigned to formation of the catalytic site and hydrolysis, respectively. Using isotopically labeled ATP, we identified specific marker bands for protein-bound ATP (1245 cm(-1)), ADP (1101 and 1205 cm(-1)), and free phosphate (1078 cm(-1)). Cleavage of the β-phosphate-γ-phosphate bond was found to be the rate-limiting step; no protein-bound phosphate intermediate was resolved.
authors.affiliation: Department of Biophysics, Faculty of Biology and Biotechnology, Ruhr-Universität Bochum, D-44780 Bochum, Germany.
authors.forename: Falk
authors.initials: F
authors.lastname: Syberg
authors.forename: Yan
authors.initials: Y
authors.lastname: Suveyzdis
authors.forename: Carsten
authors.initials: C
authors.lastname: Kötting
authors.forename: Klaus
authors.initials: K
authors.lastname: Gerwert
authors.forename: Eckhard
authors.initials: E
authors.lastname: Hofmann
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010103:Oxygen Isotopes; D010710:Phosphates / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013997:Time Factors
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; C079726:MsbA protein, Bacteria; D010103:Oxygen Isotopes; D010710:Phosphates; D000255:Adenosine Triphosphate