2 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, 1 7610-17614), 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 variou s combinations of ligands, MBPs, and membrane-associated transporters, by utilizing reconstituted proteoliposomes, right side out membrane v esicles, and intact cells. Closed forms of wild type MBP, complexed wi th maltose or maltodextrins, interacted with wild type transporter com plex to stimulate the hydrolysis of ATP by MalK ATPase located on the other side of the membrane, as shown earlier for the maltose-MBP compl ex (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 ligand ed MBPs, such as the complex containing wild type MBP and reduced, oxi dized, or cyclic maltodextrins or the complex containing the mutant MB P MalE254 and unmodified maltodextrins, did not stimulate ATP hydrolys is, suggesting that the proper interaction between the ligand-MBP comp lex and the external surface of the transporter requires the former to be in the closed conformation. However, when a mutant transporter con taining MalG511 was used, the already significant basal level of ATP h ydrolysis 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 al ways require the closed MBP complex presumably because of its exceptio nally strong affinity to MBP, described earlier (Dean, D. A., Her, L.- I., Shuman, H. A., and Nikaido, H. (1992) Mel. Microbiol. 6, 2033-2040 ). Furthermore, this mutant transporter was able to transport reduced maltodextrin, and cells expressing the transporter were able to grow b y using reduced maltodextrin, if the periplasmic concentrations of MBP were kept low so as not to inhibit the transport process.