Activation of CheY mutant D57N by phosphorylation at an alternative site, Ser‐56

Abstract

SummaryThe site of phosphorylation of the chemotaxis response regulator CheY is aspartate 57. When Asp-57 is replaced with an asparagine, the resultant protein can be phosphorylated at an alternative site. We report here that phosphorylation of this mutant protein, CheY D57N, at the alternative site affords the protein activity in vivo in the absence of CheZ. Using a direct phosphopeptide mapping approach, we identified the alternate phosphorylation site as serine 56. Introduction of a Ser→Ala substitution at this position in wild-type CheY had no effect on function. However, replacement of Ser-56 with Ala in CheY D57N abrogated the activity seen in vivo for the CheY D57N single mutant protein, and no phosphorylation of the CheY S56A/D57N double mutant protein was observed in vitro. Construction and analysis of double mutants CheY D57N/T87A and CheY D57N/K109R, which were both inactive, suggested that phosphorylation at Ser-56 or Asp-57 may activate the protein by similar mechanisms. In contrast to CheY D57N, mutant CheY D57E displayed no activity in vivo, despite its ability to be phosphorylated in vitro. Acid-base stability analysis indicated that CheY D57E phosphorylates on an acidic residue, presumably Glu-57. These data suggest that a key determinant of the ability of a phosphoryl group to activate CheY is proximity to the hydrophobic core of the protein, with consequent opportunity to reposition key residues, irrespective of the chemical nature of the linkage attaching the phosphoryl group to CheY.

Phosphorylation-Independent Activity of Atypical Response Regulators ofHelicobacter pylori

“…These results support the hypothesis that phosphorylation of S47 or S48 in the receiver domain is not required for the function of response regulator HP1021 necessary for normal cell growth. Because the receiver sequence of HP1021 lacks an amino acid with a carbonyl side chain in the vicinity of the serine residues, this result is in line with our current understanding of the phosphotransfer reaction to the receiver domain (2,25). Moreover, when whole-cell protein lysates of H. pylori were separated by two-dimensional gel electrophoresis and the spot corresponding to HP1021 was analyzed by mass spectrometry, no evidence of serine phosphorylation was obtained (data not shown).…”

“…2B). It has been reported that in a D57N mutant of E. coli CheY the neighboring serine residue (S56) is phosphorylated by the histidine kinase CheA, although the efficiency is clearly reduced, demonstrating that the highenergy phosphoramidate of the kinase can serve as a phosphoryl source to generate serine phosphate (2). Phosphorylation of a D54N mutant of NtrC from E. coli which likewise harbors a serine residue N terminally adjacent to D54 was also observed, while D54T and D54Y mutants of NtrC were not phosphorylated by the cognate histidine kinase NtrB in vitro (25).…”

“…Phosphorylation of a D54N mutant of NtrC from E. coli which likewise harbors a serine residue N terminally adjacent to D54 was also observed, while D54T and D54Y mutants of NtrC were not phosphorylated by the cognate histidine kinase NtrB in vitro (25). Therefore, it was speculated that alternative-site phosphorylation of serine requires the presence of an amino acid containing a carbonyl group in the neighboring position that acts as a ligand for Mg 2ϩ , which is important in catalyzing the phosphotransfer reaction (2). In neither HP1021 nor its orthologs from H. hepaticus, C. jejuni, and W. succinogenes is an amino acid with a carbonyl side chain located adjacent to S47 (Fig.…”

Competence Repression under Oxygen Limitation through the Two-Component MicAB Signal-Transducing System in Streptococcus pneumoniae and Involvement of the PAS Domain of MicB

“…Both phosphorylated proteins were labile in alkali and stable in acid (Fig. 6B), which indicates that an aspartate residue was likely phosphorylated in both (1,11). However, MicA59DA-PO 4 had a half-life of 12 min whereas MicA-PO 4 had a half-life of over 20 min (Fig.…”

The CheYs of Rhodobacter sphaeroides

“…A previous study on E. coli CheY has shown that the CheY(D57N) mutant is partially activated by phosphorylation of Ser-56 (39). Phosphorylation of mutants where the primary phosphorylation site has been replaced with asparagine has also been detected for the response regulators FixJ from S. meliloti and NtrC from E. coli (54,55).…”