THE RESPONSE REGULATORS CHEB AND CHEY EXHIBIT COMPETITIVE-BINDING TO THE KINASE CHEA

The autophosphorylating kinase CheA of the bacterial chemosensory signaling pathway donates a phosphoryl group to either of two response regulator proteins, CheY or the receptor methylesterase (CheB). With isothermal titration calorimetry, it was demonstrated that CheA and a CheA fragment composed of amino acid residues 1-233 (CheA(1-233)) bound to CheY with similar dissociation constants of 2.0 and 1.2 mu M at 298 K respectively, indicating that the CheY binding site is wholly within the 1-233 amino acid locus. CheB bound to CheA(1-233) With a K-D Of 3.2 mu M, and also bound to intact CheA with the same affinity. CheY was found to compete with CheB for binding to CheA(1-233), in spite of the low level of sequence identity between CheY and the regulatory domain of CheB. The competitive nature of CheY and CheB binding was determined in two independent sets of experiments: titration experiments in which either a CheB-CheA(1-233) complex was titrated with CheY or CheB was titrated with a CheY-CheA(1-233) complex, and competitive affinity chromatography experiments that used Ni-NTA-chelating resin as an affinity matrix for complexes of the histidine-tagged CheA(1-233) fragment and CheY or CheB. The effects of phosphorylation, binding-site mutations, and active-site mutations were also studied to probe the influence of conformational changes in CheY as a regulatory mechanism of CheY-CheA interactions. Phosphorylated CheY, in the presence of excess EDTA, was found to have a 2-fold lower affinity for CheA(1-233), and 6 mM Mg2+ further reduced the affinity of phosphorylated CheY for CheA(1-233) (ca. 3-fold), although Mg2+ On it, own had no effect on the interactions of either CheB or CheY with CheA(1-233) The data thus indicate that phosphorylated CheY has a significantly lower affinity for CheA under physiological conditions. The idea that phosphorylation may induce a significant conformational change, reducing the strength of the CheY-CheA interactions, is supported by the relative values of the association constants measured for CheY active-site and binding-site mutants. A binding-site mutation (A103V) in CheY, which is remote from the site of phosphorylation produced a 10-fold reduction in K-a, whereas active-site mutations produced a modest (2-fold) reduction.