Activation of Methylesterase CheB:  Evidence of a Dual Role for the Regulatory Domain

Abstract

The response regulator CheB functions within the bacterial chemotaxis system together with the methyltransferase CheR to control the level of chemoreceptor methylation, influencing the signaling activities of the receptors. CheB catalyzes demethylation of specific methylglutamate residues introduced into the chemoreceptors by CheR. CheB has a two-domain architecture consisting of an N-terminal regulatory domain joined by a linker to a C-terminal effector domain. In the unphosphorylated state of the response regulator, the regulatory domain inhibits the methylesterase activity of the effector domain. Upon phosphorylation of a specific aspartate residue within the regulatory domain, the C-terminal methylesterase activity is stimulated, resulting in the subsequent demethylation of the chemoreceptors. We have investigated the mechanism of regulation of CheB activity by the N-terminal regulatory domain. First, we have found that phosphorylation of the N-terminal domain not only relieves inhibition of the C-terminal methylesterase activity but also provides an enhancement of this activity above that seen for the C-terminal effector domain alone. Second, we have identified mutations in CheB that show an enhancement of methylesterase activity in the absence of phosphorylation. Most of these single-site mutations are localized in the linker region joining the regulatory and effector domains. On the basis of these observations, we propose a model for activation of CheB in which phosphorylation of the regulatory domain results in a reorganization of the domain interface, allowing exposure of the active site to the receptor substrate and simultaneously stimulating methylesterase activity.

The Campylobacter jejuni Response Regulator, CbrR, Modulates Sodium Deoxycholate Resistance and Chicken Colonization

“…However, it is not known whether CbrR transfers a phosphate to a partner HK or RR. It is also possible that one or both RR domains control the activity of the GGDEF domain in a manner similar to the phosphorylation of the RR domain of CheB, which, in turn, activates a C-terminal methylesterase domain (3). Alternatively, CbrR may interact directly with another protein.…”

How Signals Are Heard during Bacterial Chemotaxis: Protein-Protein Interactions in Sensory Signal Propagation

“…When this domain is not phosphorylated, it inhibits the esterase activity of CheB; when phosphorylated, it stimulates this activity (references 5, 34, and 70 and references therein). On the basis of structural and biochemical data, it was proposed that phosphorylation of the regulatory domain results in reorganization of its interface, exposing the active site to the receptor, and simultaneously stimulating the methylesterase activity of CheB (5,34). The interaction between CheB and the receptors is complex and probably involves several regions on the surface of CheB (35).…”

C-terminal DNA binding stimulates N-terminal phosphorylation of the outer membrane protein regulator OmpR from Escherichia coli

“…Because we observe stimulation of OmpR phosphorylation by both acetyl phosphate as well as the kinase EnvZ, the latter interpretation seems to be the more likely one. 44 M EnvZ115 (bottom row), as described in Materials and Methods. OmpR-P is expressed as a percentage of the total OmpR protein present in the reaction, as determined by reverse-phase HPLC on a C4 column in the absence of C1 DNA (ϪC1) and in the presence of a 2-fold molar excess of C1 DNA (ϩC1).…”

“…The two domains are connected by an 18-residue linker that, except for a short helical turn of 4 residues, does not exhibit secondary structure (43). Mutations that enhance methylesterase activity in the absence of phosphorylation have been isolated, and seven of eight of the substitutions map to this linker region (44).…”