The accessibility of cys-120 in CheA(S) is important for the binding of CheZ and enhancement of CheZ phosphatase activity.
The cheA gene of Escherichia coli encodes two proteins from in-frame tandem translation start sites. The long form of CheA (CheA(L)) is the histidine kinase responsible for phosphorylating the response regulator, CheY. The short form of CheA (CheA(S)) is identical in domain structure to CheA(L) except that it is missing the first 97 amino acids. Reduced CheA(S) bound to and enhanced the activity of the phosphatase of phospho-CheY, CheZ. Oxidized CheA(S) was unable to interact with CheZ. Oxidized CheA(S) formed covalent dimers, whereas CheA(L) did not. This property was believed to be the result of an intermolecular disulfide bond. The CheA proteins contain three cysteine residues, one of which likely lies within the CheZ binding region of CheA(S) and is exposed to solvent. We identified the CheZ binding domain of CheA(S) by testing the various fragments of CheA(S) that contain cysteine residues for CheZ binding activity in an ELISA-based CheA(S)-CheZ binding assay. Fragments of CheA(S) lacking the truncated P1 domain of CheA(S) ('P1) were unable to bind CheZ. We also found that a fusion of the first 42 amino acids of CheA(S) ('P1 domain) to GST bound CheZ and enhanced its activity. The interaction between the GST-CheA[98-139] fusion protein and CheZ was dependent on the accessibility of a cysteine residue (Cys-120) located in the 'P1 domain.
O'Connor
Christopher
C
Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Matsumura
Philip
P
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002633:Chemotaxis; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005982:Glutathione Transferase / Q000378:metabolism; D000071677:Histidine Kinase; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D011993:Recombinant Fusion Proteins; C000607606:cheY protein, E coli; D005982:Glutathione Transferase; D000071677:Histidine Kinase; C000607608:cheA protein, E coli; D003545:Cysteine