Analytical sedimentation of the IIA(Chb) and IIB(Chb) proteins of the Escherichia coli N,N'-diacetylchitobiose phosphotransferase system. Demonstration of a model phosphotransfer transition state complex

N. Keyhani, M. E. Rodgers, B. Demeler, J. C. Hansen, S. Roseman

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

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 the Escherichia 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, indicaring 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).

Original languageEnglish (US)
Pages (from-to)33110-33115
Number of pages6
JournalJournal of Biological Chemistry
Volume275
Issue number42
DOIs
StatePublished - Oct 20 2000

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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