2 MODES OF LIGAND-BINDING IN MALTOSE-BINDING PROTEIN OF ESCHERICHIA-COLI - ELECTRON-PARAMAGNETIC-RESONANCE STUDY OF LIGAND-INDUCED GLOBAL CONFORMATIONAL-CHANGES BY SITE-DIRECTED SPIN-LABELING

Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closur e of its two lobes. We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels. Using EPR spectroscopy, we examined the changes, cau sed by the Ligand binding, in distance between the two spin labels, he nce between the two lobes. The binding of both maltose and maltotetrao se induced a considerable closure of the N- and C-terminal lobes of MB P, Little closure occurred upon the binding of maltotetraitol or beta- cyclodextrin. Previous study by fluorescence and UV differential absor bance spectroscopy (Ball, J. A., Gehring, K., and Nikaido, H. (1997) J . Biol. Chem. 272, 17605-17609) showed that maltose and a large portio n of maltotetraose bound to MBP via one mode (R mode or ''end-on'' mod e), which is physiologically active and leads to the subsequent transp ort of the ligands across the cytoplasmic membrane, in contrast, malto tetraitol and beta-cyclodextrin bound to MBP via a different mode (B m ode or ''middle'' mode), which is physiologically inactive. The presen t work suggests that the B mode is nonproductive because ligands bindi ng in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interact ing properly with the inner membrane-associated transporter complex.