Expression of CheA fragments which define domains encoding kinase, phosphotransfer, and CheY binding activities.
The histidine protein kinase CheA is a central component of the Escherichia coli chemotaxis system. The autophosphorylation activity of CheA is controlled by membrane-bound chemoreceptors and by the CheW coupling protein. CheA phosphorylates the CheY and CheB proteins which respectively control the direction of flagellar rotation and the level of receptor adaptation, thereby regulating the cells' chemotactic response. Genes encoding three polypeptide fragments of CheA were constructed and expressed in order to better define the functional organization of the wild-type protein. These fragments allowed the identification of regions of the protein responsible for CheY binding, phosphotransfer, and kinase activity. The kinase domain was expressed as a 30-kDa polypeptide corresponding to the central portion of the wild-type protein which contains sequences homologous to other histidine kinases. It was able to phosphorylate a 15-kDa amino-terminal phosphotransfer domain which was separately expressed and purified. This latter domain is capable of phosphotransfer to CheY despite the fact that it lacks the ability to stably bind CheY. CheY was immobilized to a dextran matrix through a single cysteine residue which was introduced into the protein at a position far removed from the active site. A stable binding site for CheY was mapped to a segment between the site of autophosphorylation and the kinase domain by using surface plasmon resonance to detect binding to the immobilized CheY. The region of the kinase which tightly binds the unphosphorylated substrate may play an important role in regulating the specificity of the signal transducing system.
Swanson
R V
RV
Division of Biology, California Institute of Technology, Pasadena 91125.
Schuster
S C
SC
Simon
M I
MI
D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D002633:Chemotaxis; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000072236:Methyl-Accepting Chemotaxis Proteins; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010446:Peptide Fragments; C000607606:cheY protein, E coli; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli