Phosphorylation-induced signal propagation in the response regulator NtrC

J. Lee, J. T. Owens, I. Hwang, C. Meares, S. Kustu

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

The bacterial enhancer-binding protein NtrC is a well-studied response regulator in a two-component regulatory system. The amino (N)-terminal receiver domain of NtrC modulates the function of its adjacent output domain, which activates transcription by the σ54 holoenzyme. When a specific aspartate residue in the receiver domain of NtrC is phosphorylated, the dimeric protein forms an oligomer that is capable of ATP hydrolysis and transcriptional activation. A chemical protein cleavage method was used to investigate signal propagation from the phosphorylated receiver domain of NtrC, which acts positively, to its central output domain. The iron chelate reagent Fe-BABE was conjugated onto unique cysteines introduced into the N-terminal domain of NtrC, and the conjugated proteins were subjected to Fe-dependent cleavage with or without prior phosphorylation. Phosphorylation-dependent cleavage, which requires proximity and an appropriate orientation of the peptide backbone to the tethered Fe-EDTA, was particularly prominent with conjugated NtrC(D86C), in which the unique cysteine lies near the top of α-helix 4. Cleavage occurred outside the receiver domain itself and on the partner subunit of the derivatized monomer in an NtrC dimer. The results are commensurate with the hypothesis that α-helix 4 of the phosphorylated receiver domain of NtrC interacts with the beginning of the central domain for signal propagation. They imply that the phosphorylation-dependent interdomain and intermolecular interactions between the receiver domain of one subunit and the output domain of its partner subunit in an NtrC dimer precede - and may give rise to - the oligomerization needed for transcriptional activation.

Original languageEnglish (US)
Pages (from-to)5188-5195
Number of pages8
JournalJournal of Bacteriology
Volume182
Issue number18
DOIs
StatePublished - 2000

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

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