Two modes of ligand binding in maltose-binding protein of Escherichia coli. Functional significance in active transport.
In the preceding two papers (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609; Hall, J. A., Thorgeirson, T. E., Liu, J., Shin, Y.-E., and Nikaido, H. (1997) J. Biol. Chem. 272, 17610-17614), we showed that ligands that bind to the Escherichia coli maltose-binding protein (MBP) without producing the closure of its two lobes are not transported into the cytoplasm. Here, we examine various combinations of ligands, MBPs, and membrane-associated transporters, by utilizing reconstituted proteoliposomes, right side-out membrane vesicles, and intact cells. Closed forms of wild type MBP, complexed with maltose or maltodextrins, interacted with wild type transporter complex to stimulate the hydrolysis of ATP by MalK ATPase located on the other side of the membrane, as shown earlier for the maltose-MBP complex (Davidson, A. L., Shuman, H. A., and Nikaido, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2360-2364). In contrast, open forms of liganded MBPs, such as the complex containing wild type MBP and reduced, oxidized, or cyclic maltodextrins or the complex containing the mutant MBP MalE254 and unmodified maltodextrins, did not stimulate ATP hydrolysis, suggesting that the proper interaction between the ligand-MBP complex and the external surface of the transporter requires the former to be in the closed conformation. However, when a mutant transporter containing MalG511 was used, the already significant basal level of ATP hydrolysis was further stimulated not only by ligand MBPs in the closed form but also by those in the open form (except that containing beta-cyclodextrin), data suggesting that the mutant transporter does not always require the closed MBP complex presumably because of its exceptionally strong affinity to MBP, described earlier (Dean, D. A., Hor, L.-I., Shuman, H. A., and Nikaido, H. (1992) Mol. Microbiol. 6, 2033-2040). Furthermore, this mutant transporter was able to transport reduced maltodextrin, and cells expressing the transporter were able to grow by using reduced maltodextrin, if the periplasmic concentrations of MBP were kept low so as not to inhibit the transport process.
Hall
J A
JA
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
Ganesan
A K
AK
Chen
J
J
Nikaido
H
H
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000378:metabolism; D000483:Alleles; D001426:Bacterial Proteins / Q000378:metabolism*; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006868:Hydrolysis; D008024:Ligands; D008320:Maltose / Q000031:analogs & derivatives / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008956:Models, Chemical; D009004:Monosaccharide Transport Proteins; D016296:Mutagenesis; D009844:Oligosaccharides / Q000378:metabolism; D033902:Periplasmic Binding Proteins; D011134:Polysaccharides / Q000378:metabolism; D011487:Protein Conformation
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; C461619:MalE protein, E coli; C068021:MalK protein, Bacteria; C489398:MalK protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009844:Oligosaccharides; D033902:Periplasmic Binding Proteins; D011134:Polysaccharides; C461599:maltose transport system, E coli; D008320:Maltose; C008315:maltodextrin; D000255:Adenosine Triphosphate; C009819:maltotetraose