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Discrimination between different methylation states of chemotaxis receptor Tar by receptor methyltransferase CheR.

ABSTRACT: Bacterial chemotaxis receptors are posttranslationally modified by carboxyl methylation of specific glutamate residues within their cytoplasmic domains. This highly regulated, reversible modification counterbalances the signaling effects of ligand binding and contributes to adaptation. On the basis of the crystal structure of the gamma-glutamyl methyltransferase CheR, we have postulated that positively charged residues in helix alpha2 in the N-terminal domain of the enzyme may be complementary to the negatively charged methylation region of the methyltransferase substrates, the bacterial chemotaxis receptors. Several altered CheR proteins, in which positively charged arginine or lysine residues were substituted with alanines, were constructed and assayed for their methylation activities toward wild-type receptor and a series of receptor variants containing different glutamates available for methylation. One of the CheR mutant proteins (Arg53Ala) showed significantly lower activity toward all receptor constructs, suggesting that Arg53 may play a general role in catalysis of methyl transfer. The rest of the mutant proteins exhibited different patterns of relative methylation rates toward different receptor substrates, indicating specificity, probably through interaction of CheR with the receptor at sites distal to the specific site of methylation. The findings imply complementarity between positively charged residues of the alpha2 helix of CheR and the negatively charged glutamates of the receptor. It is likely that this complementarity is involved in discriminating different methylation states of the receptors.

SUBMITTER: Perez E

PROVIDER: S-EPMC3645282 | biostudies-literature | 2004 Feb

REPOSITORIES: biostudies-literature

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Discrimination between different methylation states of chemotaxis receptor Tar by receptor methyltransferase CheR.

Perez Eduardo E West Ann H AH Stock Ann M AM Djordjevic Snezana S

Biochemistry 20040201 4

Bacterial chemotaxis receptors are posttranslationally modified by carboxyl methylation of specific glutamate residues within their cytoplasmic domains. This highly regulated, reversible modification counterbalances the signaling effects of ligand binding and contributes to adaptation. On the basis of the crystal structure of the gamma-glutamyl methyltransferase CheR, we have postulated that positively charged residues in helix alpha2 in the N-terminal domain of the enzyme may be complementary t ...[more]

PMID: 14744139

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Project description:Methylation of specific chemoreceptor glutamyl residues by methyltransferase CheR mediates sensory adaptation and gradient sensing in bacterial chemotaxis. Enzyme action is a function of chemoreceptor signaling conformation: kinase-off receptors are more readily methylated than kinase-on, a feature central to adaptational and gradient-sensing mechanisms. Differential enzyme action could reflect differential binding, catalysis or both. We investigated by measuring CheR binding to kinase-off and kinase-on forms of Escherichia coli aspartate receptor Tar deleted of its CheR-tethering, carboxyl terminus pentapeptide. This allowed characterization of the low-affinity binding of enzyme to the substrate receptor body, otherwise masked by high-affinity interaction with pentapeptide. We quantified the low-affinity protein-protein interactions by determining kinetic rate constants of association and dissociation using bio-layer interferometry and from those values calculating equilibrium constants. Whether Tar signaling conformations were shifted by ligand occupancy or adaptational modification, there was little or no difference between the two signaling conformations in kinetic or equilibrium parameters of enzyme-receptor binding. Thus, differential methyltransferase action does not reflect differential binding. Instead, the predominant determinants of binding must be common to different signaling conformations. Characterization of the dependence of association rate constants on Deybe length, a measure of the influence of electrostatics, implicated electrostatic interactions as a common binding determinant. Taken together, our observations indicate that differential action of methyltransferase on kinase-off and kinase-on chemoreceptors is not the result of differential binding and suggest it reflects differential catalytic propensity. Differential catalysis rather than binding could well be central to other enzymes distinguishing alternative conformations of protein substrates.

Dataset Information

Discrimination between different methylation states of chemotaxis receptor Tar by receptor methyltransferase CheR.

Publications

Discrimination between different methylation states of chemotaxis receptor Tar by receptor methyltransferase CheR.

Similar Datasets

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