A comparative electron paramagnetic resonance study of the nucleotide-binding domains' catalytic cycle in the assembled maltose ATP-binding cassette importer.
We present a quantitative analysis of conformational changes of the nucleotide-binding subunits, MalK(2), of the maltose ATP-binding cassette importer MalFGK(2) during the transport cycle. Distance changes occurring between selected residues were monitored in the full transporter by site-directed spin-labeling electron paramagnetic resonance spectroscopy and site-directed chemical cross-linking. We considered S83C and A85C from the conserved Q-loop and V117C located on the outer surface of MalK. Additionally, two native cysteines (C350, C360) were included in the study. On ATP binding, small rearrangements between the native sites, and no distance changes between positions 117 were detected. In contrast, positions 85 come closer together in the ATP-bound state and in the vanadate-trapped intermediate and move back toward the apo-state after ATP hydrolysis. The distance between positions 83 is shown to slightly decrease on ATP binding, and to further decrease after ATP hydrolysis. Results from cross-linking experiments are in agreement with these findings. The data are compared with in silico spin-labeled x-ray structures from both isolated MalK(2) and the MalFGK(2)-E complex. Our results are consistent with a slightly modified "tweezers-like" model of closure and reopening of MalK(2) during the catalytic cycle, and show an unforeseen potential interaction between MalK and the transmembrane subunit MalG.
Grote
Mathias
M
Institut für Biologie/Bakterienphysiologie, Humboldt Universität zu Berlin, Berlin, Germany.
Bordignon
Enrica
E
Polyhach
Yevhen
Y
Jeschke
Gunnar
G
Steinhoff
Heinz-Jürgen
HJ
Schneider
Erwin
E
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000476:Alkanesulfonates / Q000378:metabolism; D001696:Biomechanical Phenomena; D002384:Catalysis; D019295:Computational Biology; D003432:Cross-Linking Reagents / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012995:Solubility; D013113:Spin Labels
D018528:ATP-Binding Cassette Transporters; D000476:Alkanesulfonates; D003432:Cross-Linking Reagents; C468279:MalFGK(2) protein, Salmonella typhimurium; D009004:Monosaccharide Transport Proteins; D009711:Nucleotides; D013113:Spin Labels; D003545:Cysteine