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

The autophosphorylating kinase CheA of the bacterial chemosensory sign aling pathway donates a phosphoryl group to either of two response reg ulator proteins, CheY or the receptor methylesterase (CheB). With isot hermal 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 th e low level of sequence identity between CheY and the regulatory domai n of CheB. The competitive nature of CheY and CheB binding was determi ned in two independent sets of experiments: titration experiments in w hich 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 aff inity matrix for complexes of the histidine-tagged CheA(1-233) fragmen t and CheY or CheB. The effects of phosphorylation, binding-site mutat ions, and active-site mutations were also studied to probe the influen ce of conformational changes in CheY as a regulatory mechanism of CheY -CheA interactions. Phosphorylated CheY, in the presence of excess EDT A, 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(1-2 33) (ca. 3-fold), although Mg2+ On it, own had no effect on the intera ctions of either CheB or CheY with CheA(1-233) The data thus indicate that phosphorylated CheY has a significantly lower affinity for CheA u nder physiological conditions. The idea that phosphorylation may induc e a significant conformational change, reducing the strength of the Ch eY-CheA interactions, is supported by the relative values of the assoc iation constants measured for CheY active-site and binding-site mutant s. A binding-site mutation (A103V) in CheY, which is remote from the s ite of phosphorylation produced a 10-fold reduction in K-a, whereas ac tive-site mutations produced a modest (2-fold) reduction.