The protein-protein interaction network of the Escherichia coli EIIA regulatory protein reveals a role in cell motility and metabolic control.

The nitrogen-related PTS system, present in many Proteobacteria including Escherichia coli, acts as a phosphorelay cascade composed of the EI, NPr and EIIA proteins. Phosphotransfer initiates with phosphoenolpyruvate-dependent EI autophosphorylation, the phosphoryl group is then transferred to NPr and finally to a conserved histidine residue on EIIA. The reporter metabolites l-glutamine and 2-oxoglutarate reciprocally regulate EI autophosphorylation (Lee et al., 2013) and consequently the phosphorylation status of the PTS components is controlled by the availability of nitrogen and carbon. The final phosphate acceptor, EIIA, regulates a range of cellular process by acting as the central hub of a complex protein-protein interaction network. Contact between EIIA and its target proteins is usually regulated by the EIIA phosphorylation status. In this study we performed ligand fishing assays coupled to label-free quantitative proteomics to examine the protein-protein interaction network of E. coli EIIA and a phosphomimic variant of the protein. The ligand fishing data, along with phenotypic analysis, indicated that EIIA interacts with proteins related to chemotaxis and thereby regulates cell motility. Important metabolic enzymes were also identified as potential EIIA binding partners.