Activation of CheY mutant D57N by phosphorylation at an alternative site, Ser-56.
The 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.
Appleby
J L
JL
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599-7290, USA.
Bourret
R B
RB
D019943:Amino Acid Substitution; D001426:Bacterial Proteins; D002633:Chemotaxis; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology; D029968:Escherichia coli Proteins; D005407:Flagella / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D000072236:Methyl-Accepting Chemotaxis Proteins; D009154:Mutation; D010449:Peptide Mapping / Q000379:methods; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D015398:Signal Transduction
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C000607606:cheY protein, E coli; D010768:Phosphoserine; D011494:Protein Kinases