Mutations leading to altered CheA binding cluster on a face of CheY.
CheY is the response regulator of Escherichia coli chemotaxis and is one of the best studied response regulators of the two-component signaling system. CheY can receive phosphate from the histidine kinase, CheA. Phospho-CheY interacts with the motor-switch complex to induce clockwise flagellar rotation, thus causing the cell to tumble. We used an enzyme-linked immunosorbent assay to study the direct interaction between the kinase, CheA, and the regulator, CheY. The products of random, suppressor, and site-specific cheY mutants were assayed for their ability to bind CheA. Nine mutants showed altered binding. We sequenced and mapped these point mutations on the crystal structure of CheY, and a high degree of spatial clustering was revealed, indicating that this region of CheY is involved in CheA binding. Interestingly, five of these altered binding mutants were previously defined as being involved in motor-switch binding interactions. This suggested a possible overlap between the motor-switch binding and CheA binding surfaces of CheY. Using CheY (Trp-58) fluorescence quenching, we determined the equilibrium dissociation constants of CheA (124-257) binding for these CheY mutants. The results from the fluorescence quenching are in close agreement with our initial enzyme-linked immunosorbent assay results. Therefore, we propose that the CheA and the motor binding surfaces on CheY partially overlap and that this overlap allows CheY to interact with either the CheA or the flagellar motor, depending on its signaling (phosphorylation) state.
Shukla
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Department of Microbiology and Immunology, University of Illinois at Chicago 60612, USA.
Matsumura
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008958:Models, Molecular; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011494:Protein Kinases / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011994:Recombinant Proteins; C000607606:cheY protein, E coli; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli