Evidence for phosphorylation-dependent conformational changes in methylesterase CheB

Enhancement of methylesterase activity of the response regulator CheB is de pendent upon phosphorylation of the N-terminal regulatory domain of the enz yme. This domain plays a dual role in the regulation of methylesterase acti vity with an inhibitory effect in the unphosphorylated state and a stimulat ory effect in the phosphorylated state. Structural studies of the unphospho rylated state have indicated that the basis for the regulatory domain's inh ibitory effect is partial blockage of access of substrate to the active sit e suggesting that the activation upon phosphorylation involves a reposition ing of the two domains with respect to each other. We report in this study evidence for phosphorylation-dependent conformational changes in CheB. Diff erences in rates of proteolytic cleavage by trypsin between the phosphoryla ted and unphosphorylated states have been observed at three sites in the pr otein with one site, 113, within the regulatory domain and two sites, 134 a nd 148, lying within the interdomain linker. These results support the hypo thesis for the: mechanism for the activation of CheB wherein phosphorylatio n of a specific apartate residue within the N-terminal domain results in a propagated conformational change within the regulatory domain leading to a repositioning of its two domains. Presumably, structural changes in the reg ulatory domain of CheB facilitate a repositioning of the N- and C-terminal domains, leading to stimulation of methylesterase activity.