The Accessibility of Cys-120 in CheA_S Is Important for the Binding of CheZ and Enhancement of CheZ Phosphatase Activity

摘要

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 (CheAs) is identical in domain structure to CheA_L except that it is missing the first 97 amino acids.Reduced CheAs bound to and enhanced the activity of the phosphatase of phospho-CheY,CheZ.Oxidized CheAs was unable to interact with CheZ.Oxidized CheAs formed covalent dimers,whereas CheAL 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 CheAs and is exposed to solvent.We identified the CheZ binding domain of CheAs by testing the various fragments of CheAs that contain cysteine residues for CheZ binding activity in an ELISA-based CheAs-CheZ binding assay.Fragments of CheAs lacking the truncated P1 domain of CheAs ('P1) were unable to bind CheZ.We also found that a fusion of the first 42 amino acids of CheAs ('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.