Ligand-Specific Activation of Escherichia coli Chemoreceptor Transmethylation

Abstract

Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by the enzymecatalyzed methylation (and demethylation) of glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, where the methyltransferase (CheR) molecule catalyzing methyl group transfer is tethered to the C terminus of a neighboring receptor. Here, it was shown that E. coli cells exhibited adaptation to attractant stimuli mediated through either engineered or naturally occurring MCPs that were unable to tether CheR as long as another MCP capable of tethering CheR was also present, e.g., either the full-length aspartate or serine receptor (Tar or Tsr). Methylation of isolated membrane samples in which engineered tethering and substrate receptors were coexpressed demonstrated that the truncated substrate receptors (trTsr) were efficiently methylated in the presence of tethering receptors (Tar with methylation sites blocked) relative to samples in which none of the MCPs had tethering sites. The effects of ligand binding on methylation were investigated, and an increase in rate was produced only with serine (the ligand specific for the substrate receptor trTsr); no significant change in rate was produced by aspartate (the ligand specific for the tethering receptor Tar). Although the overall efficiency of methylation was lower, receptor-specific effects were also observed in trTar-and trTsr-containing samples, where neither Tar nor Tsr possessed the CheR binding site at the C terminus. Altogether, the results are consistent with a ligand-induced conformational change that is limited to the methylated receptor dimer and does not spread to adjacent receptor dimers.Bacterial chemotaxis signaling pathways are two-component systems in which the sensors are composed of noncovalent ternary complexes of transmembrane receptor proteins, the cytoplasmic adaptor protein CheW and the histidine kinase CheA (9, 35). The transmembrane receptors, which are also known as methyl-accepting chemotaxis proteins (MCPs), are organized as homodimers to bind ligand (28), where each subunit of the dimer consists of a periplasmic ligand-binding domain (27, 49), two ␣-helical transmembrane segments (31), and a helical cytoplasmic region that contains the highly conserved signaling domain flanked by the methylation helices (16,20). Receptor dimers of different ligand specificity cluster in the membrane in synergy with CheW and CheA, frequently at the poles of the cell (26). The available evidence suggests that the receptors in these signaling patches possess the trimer-ofdimers organization, first identified in the crystal structure of the cytoplasmic domain (1, 16, 41). The complexity of the organization is further heightened by the possible involvement of interdigitating cytoplasmic domains, which have been observed by an electron microscope study of receptor arrays (46). Evidence consistent with extensive interactions among receptor dimers in the excitation and adaptation phases of sig...

Physical Responses of Bacterial Chemoreceptors

“…Since the YFPs are tethered to the receptors via a chain of several amino acids, they should be able to reorient on a nanosecond time scale. Evidently, when CheR (or CheB), which is larger then YFP, binds to a receptor at the same position, it is free to move and act on the adaptation sites of neighbouring dimers 34,35 . If the rotational diffusion of YFP were restricted, the anisotropy level in the absence of homo-FRET would be expected to exceed that of free YFP 28 .…”

Self-assembly of receptor/signaling complexes in bacterial chemotaxis

“…Additionally, conformational changes in the receptors seem to be able to inactivate CheA without requiring dissociation (24,29,38). The receptors outside the active receptor͞signaling complexes may still play an important role in cellular memory and adaptation, because these receptors would be substrates for attractant-mediated methylation and demethylation by CheR and CheB (39)(40)(41)(42). Over longer time scales, the changing methylation states of the receptors could determine which receptors participate in the active receptor͞signaling complexes.…”

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

“…4B), because attractant binding to receptors has been observed to increase the physical separation among receptors (23,24). Attractant binding also produces well known biochemical effects: a reduction in kinase activity, a rise in the methylation rate, and a lowering of the demethylation rate (22,35,39). These effects set in motion the return toward the prestimulus kinase activity (adaptation).…”