Analytical Sedimentation of the IIA^Chb and IIB^Chb Proteins of the Escherichia coli N,N′-Diacetylchitobiose Phosphotransferase System

The phosphoenolpyruvate:glycose transferase system (PTS) is a prototypic signaling system responsible for the vectorial uptake and phosphorylation of carbohydrate substrates. The accompanying papers describe the proteins and product of theEscherichia coli N,N-diacetylchitobiose ((GlcNAc)₂) PTS-mediated permease. Unlike most PTS transporters, the Chb system is composed of two soluble proteins, IIA^Chb and IIB^Chb, and one transmembrane receptor (IIC^Chb). The oligomeric states of PTS permease proteins and phosphoproteins have been difficult to determine. Using analytical ultracentrifugation, both dephospho and phosphorylated IIA^Chb are shown to exist as stable dimers, whereas IIB^Chb, phospho-IIB^Chb and the mutant Cys10SerIIB^Chb are monomers. The mutant protein Cys10SerIIB^Chb is unable to accept phosphate from phospho-IIA^Chb but forms a stable higher order complex with phospho-IIA^Chb (but not with dephospho-IIA^Chb). The stoichiometry of proteins in the purified complex was determined to be 1:1, indicating that two molecules of Cys10SerIIB^Chb are associated with one phospho-IIA^Chb dimer in the complex. The complex appears to be a transition state analogue in the phosphotransfer reaction between the proteins. A model is presented that describes the concerted assembly and disassembly of IIA^Chb-IIB^Chb complexes contingent on phosphorylation-dependent conformational changes, especially of IIA^Chb.