Structural and regulatory studies on protein components of the Escherichia coli (E. coli) sugar transport system known as the phosphoenolpyruvate: sugar phosphotransferase system (PTS) continued. The first component of the PTS (enzyme I, EI) is phosphorylated by phosphoenolpyruvate on an active site histidine and that phosphoryl group can be transferred to the active site of the second component (HPr). Previous studies have led to the elucidation of the three- dimensional structures of both the amino-terminal domain of EI (EIN) and HPr. The new studies were designed to describe, by NMR spectroscopy, the nature of the interface between EIN and HPr when the two proteins form a complex. The complex between EIN and HPr is a classical example of surface complementarity, involving an essentially all helical interface, comprising helices 2, 2, 3 and 4 of the alpha- subdomain of EIN and helices 1 and 2 of HPr, that requires virtually no changes in conformation of the components relative to that in their respective free states. The specificity of the complex is dependent on the correct placement of both van der Waals and electrostatic contacts. The transition state can be formed with minimal changes in overall conformation, and is stabilized in favor of phosphorylated HPr, thereby accounting for the directionality of phosphoryl transfer. EI undergoes a slow monomer-dimer transition. In vitro autophosphorylation of EI was studied at limiting concentrations of EI. Addition to incubation mixtures containing wild-type EI of inactive or low-activity mutant forms of EI resulted in stimulation of autophosphorylation activity. The kinetics of the activation fit well to a model in which the active form of EI is the dimer. These experiments provide support for the argument that only the dimeric form of EI can be autophosphorylated. One of the PTS proteins that can accept a phosphoryl group from P-HPr is named IIAglc. This protein also plays a role in the regulation of activity of other sugar transport systems in E. coli. By using a previously described direct binding assay, a collection of single-Cys replacement mutants in cytoplasmic loops of lactose permease was evaluated for their capacity to bind IIAglc. 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 IIAglc provides the basis for developing two regions of consensus sequence in those partner proteins necessary for binding to IIAglc. The requirement for two interaction regions is interpreted in the regulatory framework of a substrate- dependent conformational change that brings these two regions into an orientation optimal for binding IIAglc. - E. coli, sugar transport, PTS, NMR, protein structure, EI dimerization, lactose permease-IIAglc complex
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