Efficient adaptational demethylation of chemoreceptors requires the same enzyme-docking site as efficient methylation

Abstract

The mechanistic basis of sensory adaptation and gradient sensing in bacterial chemotaxis is reversible covalent modification of transmembrane chemoreceptors, methylation, and demethylation at specific glutamyl residues in their cytoplasmic domains. These reactions are catalyzed by a dedicated methyltransferase CheR and a dedicated methylesterase CheB. The esterase is also a deamidase that creates certain methyl-accepting glutamyls by hydrolysis of glutamine side chains. We investigated the action of CheB and its activated form, phospho-CheB, on a truncated form of the aspartate receptor of Escherichia coli that was missing the last 5 aa of the intact receptor. The deleted pentapeptide is conserved in several chemoreceptors in enteric and related bacteria. The truncated receptor was much less efficiently demethylated and deamidated than intact receptor, but essentially was unperturbed for kinase activation or transmembrane signaling. CheB bound specifically to an affinity column carrying the isolated pentapeptide, implying that in the intact receptor the pentapeptide serves as a docking site for the methylesterase/deamidase and that the truncated receptor was inefficiently modified because the enzyme could not dock. It is striking that the same pentapeptide serves as an activity-enhancing docking site for the methyltransferase CheR, the other enzyme involved in adaptational covalent modification of chemoreceptors. A shared docking site raises the tantalizing possibility that relative rates of methylation and demethylation could be influenced by competition between the two enzymes at that site.

Figure 1

Methyl-accepting activities of Tar and TarΔpp. Membranes providing ≈5 μM Tar or TarΔpp were incubated for 1 min at room temperature with 50 μM [methyl-³H]AdoMet, 1 mM aspartate (where indicated), and a cell extract providing ≈1 μM CheR. Samples containing ≈50 pmol receptor were analyzed for radiolabeled methyl groups. The data are averages of experiments using two independent membrane preparations. Error bars show SEs.

Figure 2

Kinase activation by unoccupied and ligand-occupied forms of Tar and TarΔpp. Membranes providing ≈2 μM Tar or TarΔpp were incubated with 0.25 μM CheA, 4 μM CheW, and 10 μM CheY to allow complex formation, ³²P-ATP was added, samples were taken 10 s later, and ³²P-labeled phospho-CheY was determined by SDS/PAGE and PhosphorImaging. Aspartate (1 mM) was present where indicated. Replicates and error bars are as for Fig. 1.

Figure 3

In vitro treatment to methylate Tar and TarΔpp increased kinase activation and preserved control by ligand occupancy. Membranes containing Tar or TarΔpp were incubated in the absence (methylation −) or presence (methylation +) of [methyl-³H]AdoMet and a CheR-containing extract. The latter condition resulted in ≈0.4 methyl groups per receptor. Kinase activation by washed, reisolated, receptor-containing membranes in the absence (−) or presence (+) of 1 mM aspartate was determined as in Fig. 2. Levels of phospho-CheY were normalized to the value for Tar in the absence of methylation and aspartate.

Figure 4

Demethylation of Tar and TarΔpp catalyzed by CheB and phospho-CheB. (A) Action of CheB on receptors alone. Membranes providing ≈10 μM Tar or TarΔpp modified with ³H-methyl groups as described for Fig. 3 (≈0.4 methyls per receptor) were mixed with 0.5 μM purified CheB. Samples containing ≈40 pmol receptor were removed at the indicated times and analyzed for released ³H-methanol. (B) Action of CheB and phospho-CheB on receptors alone. Receptor-containing membranes as in A were incubated 15 min with 5 μM CheA, 1 mM ATP was added to some incubations (plots labeled CheB-P), the incubation continued for 15 min, purified CheB was added to 0.5 μM, and samples were taken and analyzed as for A. (C) Action of CheB and phospho-CheB on receptors complexed with CheA and CheW. Receptor-containing membranes as in A were incubated 30 min with 5 μM CheA and 5 μM CheW. CheB or CheB plus ATP (plots labeled CheB-P) were added to 0.5 μM CheB and 1 mM ATP, and samples were taken and analyzed as for A. For all panels, data are averages of experiments on at least three separate membrane preparations. Error bars show SEs.

Figure 5

Deamidation of Tar and TarΔpp catalyzed by CheB. Membranes providing ≈10 μM Tar or TarΔpp were incubated in TEDG plus 10 mM MgCl₂ with 0.5 μM purified CheB. Samples containing ≈3 pmol receptor were removed at the indicated times and analyzed by SDS/PAGE and immunoblotting. Shown is the relevant portion of the blot, including the positions of unmodified (unmod.) and deamidated (deamid.) receptor. Unmodified TarΔpp migrates slightly more rapidly than unmodified Tar, consistent with a difference of five residues.

Figure 6

Retention of modification enzymes by immobilized pentapeptide and elution by free pentapeptide. Soluble lysate (ly) from cells containing CheR (A) or CheB (B) produced from an induced gene located on a multicopy plasmid was applied to a 1-ml column of resin carrying the immobilized pentapeptide, NWETF. Fractions collected during application of the sample (fl), two column volumes of buffer (bf), and three column volumes of buffer containing 5 mg/ml free pentapeptide (pp) were analyzed by SDS/PAGE. The positions of CheR (≈33 kDa) and CheB (≈37 kDa) are indicated on the respective gels. Analysis of the 37-kDa band eluted by pentapeptide in the experiment of B revealed an amino-terminal sequence (seven residues) identical to authentic CheB. Essentially identical results were obtained by using heptapeptides representing the carboxyl-terminal residues of Tsr (EENWETF) or Tar (DPNWETF) as the immobilized or eluting peptide.