Requirements for efficient methylation of Escherichia coli chemoreceptors

Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by methylation and demethylation of specific glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, in which the methyltransferase (CheR) is tethered to the C-terminus of adjacent receptors. This interaction allows receptors adapt by assistance of adjacent receptors. Since there is evidence of the extensive receptor-receptor interactions occurring in the membrane, we investigated the extent to which ligand stimulation of receptors tethering CheR affects the rate of methylation of receptors that are not involved in tethering interactions. These studies involved isolation of membranes containing coexpressed receptors of different ligand specificity, in which the CheR docking site of Tsr was eliminated (trTsr, substrate receptor) and the methylation sites of Tar were blocked (tethering receptor). Our results demonstrate that the substrate receptors (trTsr) were efficiently methylated in the presence of tethering receptors. The effects of ligand binding on methylation were investigated, and an increase in rate was produced only with the ligand specific for the substrate receptor; no significant change in rate was produced by the ligand specific for the tethering receptor. These receptor-specific effects were also observed in samples that contained CheW, which served to increase the overall methylation rate, and in trTar and trTsr containing samples, where neither Tar nor Tsr possessed a CheR binding site at the C-terminus. Our results are consistent with a ligand-induced conformational change that is limited to the receptor dimer accepting methyl groups, and does not spread to adjacent receptor dimers. Here, it is also shown a new approach in which mass spectrometry was used to determine the level of methylation of chemotaxis receptors. A simple method in which tryptic fragments were analyzed by ESI-MS/MS, without a separation step, allow us to determine the methylated sites in soluble constructs and the full-length receptors. This new approach is a challenge in the characterization of post-translational modifications, since the methyl-accepting chemotaxis proteins can be easily identified. The results support the promise of mass spectrometry as an efficient and sensitive set of approaches to determine the level and distribution of methyl groups on receptors, in the cell under physiological conditions