In vitro interaction between components of the inner membrane complex of the maltose ABC transporter of Escherichia coli: modulation by ATP.
Interactions between domains of ATP-binding cassette (ABC) transporters are of great functional importance and yet are poorly understood. To gain further knowledge of these protein-protein interactions, we studied the inner membrane complex of the maltose transporter of Escherichia coli. We focused on interactions between the nucleotide-binding protein, MalK, and the transmembrane proteins, MalF and MalG. We incubated purified MalK with inverted membrane vesicles containing MalF and MalG. MalK bound specifically to MalF and MalG and reconstituted a functional complex. We used this approach and limited proteolysis with trypsin to show that binding and hydrolysis of ATP, inducing conformational changes in MalK, modulate its interaction with MalF and MalG. MalK in the reconstituted complex was less sensitive to protease added from the cytoplasmic side of the membrane, and one proteolytic cleavage site located in the middle of a putative helical domain of MalK was protected. These results suggest that the putative helical domain of the nucleotide-binding domains is involved, through its conformational changes, in the coupling between the transmembrane domains and ATP binding/hydrolysis at the nucleotide-binding domains.
Mourez
M
M
Unité de Programmation Moléculaire et Toxicologie Génétique, CNRS URA 1444, Institut Pasteur, 25, rue du Dr Roux, F75645 Paris Cedex 15, France.
Jéhanno
M
M
Schneider
E
E
Dassa
E
E
D018528:ATP-Binding Cassette Transporters / Q000187:drug effects / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000187:drug effects / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008320:Maltose / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D014357:Trypsin / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C079123:MalG protein, E coli; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; C461599:maltose transport system, E coli; D008320:Maltose; D000255:Adenosine Triphosphate; D014357:Trypsin; D008274:Magnesium