Binding of enzyme IIAGlc, a component of the phosphoenolpyruvate:sugar phosphotransferase system, to the Escherichia coli lactose permease.
Enzyme IIA(Glc), encoded by the crr gene of the phosphoenolpyruvate:sugar phosphotransferase system, plays an important role in regulating intermediary metabolism in Escherichia coli ("catabolite repression"). One function involves inhibition of inducible transport systems ("inducer exclusion"), and with lactose permease, a galactoside is required for unphosphorylated IIA(Glc) binding to cytoplasmic loops IV/V and VI/VII [Sondej, M., Sun, J. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530]. With inside-out membrane vesicles containing the permease, [(125)I]IIA(Glc) binding promoted by melibiose exhibits an affinity (K(D)(IIA)) of approximately 1 microM and a stoichiometry of one mole of IIA(Glc) per six moles of lactose permease. Both the quantity of [(125)I]IIA(Glc) bound and the sugar concentration required for half-maximal IIA(Glc) binding (K(0.5)(IIA)(sug)) was measured for eight permease substrates. Differences in maximal IIA(Glc) binding are observed, and the K(0.5)(IIA)(sug) does not correlate with the affinity of LacY for sugar. Furthermore, K(0.5)(IIA)(sug) does not correlate with sugar affinities for various permease mutants. IIA(Glc) does not bind to a mutant (Cys154 --> Gly), which is locked in an outwardly facing conformation, binds with increased stoichiometry to mutant Lys131 --> Cys, and binds only weakly to two other mutants which appear to be predominantly in either an outwardly or an inwardly facing conformation. When the latter two mutations are combined, sugar-dependent IIA(Glc) binding returns to near wild-type levels. The findings suggest that binding of various substrates to lactose permease results in a collection of unique conformations, each of which presents a specific surface toward the inner face of the membrane that can interact to varying degrees with IIA(Glc).
Sondej
Melissa
M
Howard Hughes Medical Institute, Department of Physiology, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
Weinglass
Adam B
AB
Peterkofsky
Alan
A
Kaback
H Ronald
HR
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D015151:Immunoblotting; D007700:Kinetics; D008553:Melibiose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011869:Radioligand Assay; D011887:Raffinose / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C032242:crr protein, E coli; C021499:lactose permease; D008553:Melibiose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; D011887:Raffinose