Uncoupling substrate transport from ATP hydrolysis in the Escherichia coli maltose transporter.
Members of the ATP-binding cassette superfamily couple the energy from ATP hydrolysis to the active transport of substrates across the membrane. The maltose transporter, a well characterized model system, consists of a periplasmic maltose-binding protein (MBP) and a multisubunit membrane transporter, MalFGK(2). On the basis of the structure of the MBP-MalFGK(2) complex in an outward-facing conformation (Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L., and Chen, J. (2007) Nature 450, 515-521), we identified two mutants in transmembrane domains MalF and MalG that generated futile cycling; although interaction with MBP stimulated the ATPase activity of the transporter, maltose was not transported. Both mutants appeared to disrupt the normal transfer of maltose from MBP to MalFGK(2). In the first case, substitution of aspartate for glycine in the maltose-binding site of MalF likely generated a futile cycle by preventing maltose from binding to MalFGK(2) during the catalytic cycle. In the second case, a four-residue deletion of a periplasmic loop of MalG limited its reach into the maltose-binding pocket of MBP, allowing maltose to remain associated with MBP during the catalytic cycle. Retention of maltose in the MBP binding site in the deletion mutant, as well as insertion of this loop into the binding site in the wild type, was detected by EPR as a change in mobility of a nitroxide spin label positioned near the maltose-binding pocket of MBP.
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
Jinming
J
Cui
Sabiha
S
Qasim
Amy L
AL
Davidson
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport / Q000502:physiology; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; C461639:MalF protein, E coli; C079123:MalG protein, E coli; D009004:Monosaccharide Transport Proteins; C461599:maltose transport system, E coli