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. Based on the crystal structure of the γ-glutamyl-methyltransferase CheR, we have postulated that positively charged residues in helix α2 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 α2 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.In enteric bacteria, chemotaxis is mediated by several homologous transmembrane receptors that sense changes in concentrations of attractant and repellent molecules (reviewed in refs. (1-4)). Ligand binding to the periplasmic domains of the receptors generates conformational signals that are transmitted to the cytoplasmic domains that regulate an associated twocomponent phosphotransfer signal transduction system. Alterations in ligand concentration evoke a cellular response involving a change in direction of flagellar rotation. Following a transient response, cells return to pre-stimulus behavior, a process known as adaptation. Adaptation is in part mediated by reversible covalent modification of the chemoreceptors. Changes in receptor ligand concentration are accompanied by changes in the level of methylation at several specific glutamate residues in the cytoplasmic domains of the † A

Identification of Methylation Sites in Thermotoga maritima Chemotaxis Receptors

“…Initial rates of methylation were determined with saturating concentrations of receptors using concentrations of T. maritima CheR at which rates were linearly proportional to enzyme concentration ( Table 1). The data indicate that T. maritima receptors can be efficiently methylated in vitro at rates comparable to that previously reported for S. enterica receptor methylation at 30°C (1.1 mol CH 3 · mol CheR Ϫ1 · min Ϫ1 [24]). As expected, for all substrates, the methylation rates for both full-length receptors and cytoplasmic domains increased as the temperature was increased up to 70°C.…”

Evolutionary genomics reveals conserved structural determinants of signaling and adaptation in microbial chemoreceptors

“…Structurally, the Glx pair and small residues in the motif lie in adjacent turns on the solvent-exposed side of a methylation helix. One residue of the Glx pair is the target for methylation by CheR and for deamidation and demethylation by CheB and CheD, a receptor-modifying deamidase lacking from E. coli but typical of many chemotaxis systems (29), whereas the flanking small residues are thought to be important for correct docking at the helix by the adaptation enzymes (30,31). We collected sequences from each signaling class containing the methylation subdomain, where a Glx pair was identified in the bc heptad registers predicted to be on the solvent-exposed surface of the four-helix bundle and determined the consensus methylation sequence for each class.…”

In silico and proteomic analysis of protein methyltransferase CheR from Bacillus subtilis

“…1E). negatively charged methylation site [45]. Intriguingly, sequence analysis revealed that these residues were also conserved in BsCheR (as Lys23 and Arg30) ( Fig.…”

“…2C), have shown to be involved in the substrate recognition and binding to the methylation site [45]. Studies on CheR of S. typhimurium established the involvement of Arg53 in methyl transfer reaction, and its location in the active site, with the methylation sequence of the receptor that is oriented antiparallel to α2 helix of CheR [45]. Arg53 may stabilize or position methylatable γ-carboxlate of glutamate by ion-pair, salt-bridge A c c e p t e d M a n u s c r i p t or other electrostatic interaction.…”