Chemotactic response regulator mutant CheY95IV exhibits enhanced binding to the flagellar switch and phosphorylation-dependent constitutive signalling.
CheY, a response regulator protein in bacterial chemotaxis, mediates swimming behaviour through interaction with the flagellar switch protein, FliM. In its active, phosphorylated state, CheY binds to the motor switch complex and induces a change from counterclockwise (CCW) to clockwise (CW) flagellar rotation. The conformation of a conserved aromatic residue, tyrosine 106, has been proposed to play an important role in this signalling process. Here, we show that an isoleucine to valine substitution in CheY at position 95--in close proximity to residue 106--results in an extremely CW, hyperactive phenotype that is dependent on phosphorylation. Further biochemical characterization of this mutant protein revealed phosphorylation and dephosphorylation rates that were indistinguishable from those of wild-type CheY. CheY95IV, however, exhibited an increased binding affinity to FliM. Taken together, these results show for the first time a correlation between enhanced switch binding and constitutive signalling in bacterial chemotaxis. Considering present structural information, we also propose possible models for the role of residue 95 in the mechanism of CheY signal transduction.
Schuster
M
M
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599-7290, USA.
Abouhamad
W N
WN
Silversmith
R E
RE
Bourret
R B
RB
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002633:Chemotaxis; D005453:Fluorescence; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008958:Models, Molecular; D010766:Phosphorylation; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D015398:Signal Transduction
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C066821:FliM protein, Bacteria