Topography of the surface of the Escherichia coli phosphotransferase system protein enzyme IIA(glc) that interacts with lactose permease

The unphosphorylated form of enzyme IIA(glc) of the Escherichia coli phosph oenolpyruvate: sugar phosphotransferase system inhibits transport catalyzed by lactose permease. We (Seok et al. (1997) Proc. Natl. Acad. Sci, U.S.A. 94, 13515-13519) previously characterized the area on the cytoplasmic face of lactose permease that interacts with enzyme IIA(glc), using radioactive enzyme IIA(glc). Subsequent studies (Sondej et al. (1999) Proc. Natl. Acad. Sci, U.S.A. 96, 3525-3530) suggested consensus binding sequences on protei ns that interact with enzyme IIA(glc). The present study characterizes a re gion on the surface of enzyme IIA(glc) that interfaces with lactose permeas e. Acetylation of lysine residues by sulfosuccinimidyl acetate treatment of enzyme IIA(glc), but not lactose permease, reduced the degree of interacti on between the two proteins. To localize the lysine residue(s) on enzyme II A(glc) that is(are) involved in the regulatory interaction, selected lysine residues were mutagenized. Conversion of nine separate lysines to glutamic acid resulted in proteins that were still capable of phosphoryl acceptance from HPr. Except for Lys69, all the modified proteins were as effective as the wild-type enzyme IIA(glc) in a test for binding to lactose permease, T he Lys69 mutant was also defective in phosphoryl transfer to glucose permea se. To derive further information concerning the contact surface, additiona l selected residues in the vicinity of Lys69 were mutagenized and tested fo r binding to lactose permease. On the basis of these studies, a model for t he region of the surface of enzyme IIAp(glc) that interacts with lactose pe rmease is proposed.