Deduction of consensus binding sequences on proteins that bind IIA(Glc) of the phosphoenolpyruvate:sugar phosphotransferase system by cysteine scanning mutagenesis of Escherichia coli lactose permease

Mediated by the protein IIA(Glc), the phosphoenolpyruvate:sugar phosphotransferase system plays a role in the regulation of activity of other sugar transport systems in Escherichia coli. By using a direct binding assay, a collection of single-Cys replacement mutants in cytoplasmic loops of lactose permease were evaluated for their capacity to bind IIA(Glc). Selected Cys replacements in loops IV/V or VI/VII result in loss of binding activity. Analysis of the mutagenesis results together with multiple sequence alignments of a family of proteins that interacts with IIA(Glc) provides the basis for developing two regions of consensus sequence in those partner proteins necessary for binding to IIA(Glc). The requirement for two interaction regions is interpreted in the regulatory framework of a substrate-dependent conformational change that brings those two regions into an orientation optimal for binding IIA(Glc).