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G S Anand

Publications and source records attributed to G S Anand.

6 recordsLinked to original sources
PubMed2001

Synthesis and biochemical characterization of a phosphorylated analogue of the response regulator CheB.

CheB is a response regulator protein in the bacterial chemotaxis two-component signal transduction pathway. Methylesterase CheB functions together with methyltransferase CheR to modulate the level of glutamate methylation in transmembrane chemoreceptors in response to environmental stimuli. The level of glutamate methylation in turn indirectly controls the direction of flagellar rotation. Like most two-component response regulators, CheB is activated in vivo by phosphorylation of a single aspartate, Asp 56, in its regulatory domain. Extensive biochemical and crystallographic studies have been completed on the inactive, unphosphorylated form of CheB. Because of the inherent lability of aspartyl phosphate bonds and the intrinsic phosphatase activity of CheB, the activated, phosphorylated form of CheB cannot be isolated for further characterization. We present a synthetic scheme to prepare an analogue of phosphorylated CheB using site-specific mutagenesis and chemical modification strategies. Initially, the two native cysteines found in CheB were substituted by serines and a cysteine was substituted for Asp 56 to yield D56C/C207S/C309S CheB. The unique cysteine in the substituted form of CheB was modified by sodium thiophosphate, Na(3)SPO(3), using two sequential disulfide bond exchange reactions. The analogue, D56C/C207S/C309S CheB-SPO(3), contained a thiophosphate group covalently bonded to the protein through a disulfide linkage at residue 56. Mass spectrometry showed that the protein was singly modified. Reverse phase chromatography showed that greater than 95% of the protein was modified under optimized conditions and that the analogue had a half-life of 28 days. In in vitro methylesterase assays in the presence of Mg(2+), the analogue exhibited activity equivalent to that of fully phosphorylated C207S/C309S CheB. Thus, D56C/C207S/C309S CheB-SPO(3) is a stable analogue that may be useful for characterization of the active form of CheB.

Aspartic Acid↗
PubMed2001

Phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of methylesterase CheB.

The crystal structure of the unphosphorylated state of methylesterase CheB shows that the regulatory domain blocks access of substrate to the active site of the catalytic domain. Phosphorylation of CheB at Asp56 results in a catalytically active transiently phosphorylated enzyme with a lifetime of approximately two seconds. Solvent accessibility changes in this transiently phosphorylated state were probed by MALDI-TOF-detected amide hydrogen/deuterium exchange. No changes in solvent accessibility were seen in the regulatory domain upon phosphorylation of Asp56, but two regions in the catalytic domain (199-203 and 310-317) became more solvent accessible. These two regions flank the active site and contain domain-domain contact residues. Comparison with results from the isolated catalytic domain-containing C-terminal fragment of CheB (residues 147-349) showed that the increased solvent accessibility was less than would have occurred upon detachment of the regulatory domain. Thus, phosphorylation causes subtle changes in solvent accessibility at the interdomain interface of CheB.

Amides↗
PubMed2000

Evidence for phosphorylation-dependent conformational changes in methylesterase CheB.

Enhancement of methylesterase activity of the response regulator CheB is dependent upon phosphorylation of the N-terminal regulatory domain of the enzyme. This domain plays a dual role in the regulation of methylesterase activity with an inhibitory effect in the unphosphorylated state and a stimulatory effect in the phosphorylated state. Structural studies of the unphosphorylated state have indicated that the basis for the regulatory domain's inhibitory effect is partial blockage of access of substrate to the active site suggesting that the activation upon phosphorylation involves a repositioning of the two domains with respect to each other. We report in this study evidence for phosphorylation-dependent conformational changes in CheB. Differences in rates of proteolytic cleavage by trypsin between the phosphorylated and unphosphorylated states have been observed at three sites in the protein with one site, 113, within the regulatory domain and two sites, 134 and 148, lying within the interdomain linker. These results support the hypothesis for the mechanism for the activation of CheB wherein phosphorylation of a specific aspartate 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 regulatory domain of CheB facilitate a repositioning of the N- and C-terminal domains, leading to stimulation of methylesterase activity.

Binding Sites↗
PubMed1998

Activation of methylesterase CheB: evidence of a dual role for the regulatory domain.

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.

Bacterial Proteins↗