Conformational coupling in the chemotaxis response regulator CheY.
CheY, a response regulator protein in bacterial chemotaxis, serves as a prototype for the analysis of response regulator function in two-component signal transduction. Phosphorylation of a conserved aspartate at the active site mediates a conformational change at a distal signaling surface that modulates interactions with the flagellar motor component FliM, the sensor kinase CheA, and the phosphatase CheZ. The objective of this study was to probe the conformational coupling between the phosphorylation site and the signaling surface of CheY in the reverse direction by quantifying phosphorylation activity in the presence and absence of peptides of CheA, CheZ, and FliM that specifically interact with CheY. Binding of these peptides dramatically impacted autophosphorylation of CheY by small molecule phosphodonors, which is indicative of reverse signal propagation in CheY. Autodephosphorylation and substrate affinity, however, were not significantly affected. Kinetic characterization of several CheY mutants suggested that conserved residues Thr-87, Tyr-106, and Lys-109, implicated in the activation mechanism, are not essential for conformational coupling. These findings provide structural and conceptual insights into the mechanism of CheY activation. Our results are consistent with a multistate thermodynamic model of response regulator activation.
Schuster
M
M
Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599-7290, USA.
Silversmith
R E
RE
Bourret
R B
RB
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002633:Chemotaxis / Q000502:physiology*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; C066821:FliM protein, Bacteria