Phosphorylation-induced signal propagation in the response regulator ntrC.

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 sigma(54) 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 alpha-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 alpha-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.