Two variable active site residues modulate response regulator phosphoryl group stability.
Many signal transduction networks control their output by switching regulatory elements on or off. To synchronize biological response with environmental stimulus, switching kinetics must be faster than changes in input. Two-component regulatory systems (used for signal transduction by bacteria, archaea and eukaryotes) switch via phosphorylation or dephosphorylation of the receiver domain in response regulator proteins. Although receiver domains share conserved active site residues and similar three-dimensional structures, rates of self-catalysed dephosphorylation span a >or= 40,000-fold range in response regulators that control diverse biological processes. For example, autodephosphorylation of the chemotaxis response regulator CheY is 640-fold faster than Spo0F, which controls sporulation. Here we demonstrate that substitutions at two variable active site positions decreased CheY autodephosphorylation up to 40-fold and increased the Spo0F rate up to 110-fold. Particular amino acids had qualitatively similar effects in different response regulators. However, mutant proteins matched to other response regulators at the two key variable positions did not always exhibit similar autodephosphorylation kinetics. Therefore, unknown factors also influence absolute rates. Understanding the effects that particular active site amino acid compositions have on autodephosphorylation rate may allow manipulation of phosphoryl group stability for useful purposes, as well as prediction of signal transduction kinetics from amino acid sequence.
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599-7290, USA.
Stephanie A
SA
Thomas
Jocelyn A
JA
Brewster
Robert B
RB
Bourret
D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010755:Organophosphates / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010755:Organophosphates; C080951:Spo0F protein, Bacillus subtilis; C011632:acetyl phosphate