Similarities and differences in interactions of the activity-enhancing chemoreceptor pentapeptide with the two enzymes of adaptational modification

Abstract

Sensory adaptation and chemotaxis by Escherichia coli require a specific pentapeptide at the chemoreceptor carboxyl terminus. This sequence binds the two enzymes of receptor adaptational modification, enhancing catalysis, but with different binding features and mechanisms. We investigated the relative importance of each pentapeptide side chain for the two enhancing interactions.

FIG. 1.

In vitro methylation and demethylation. Bars are mean values of initial rates (three or more independent experiments), normalized to wild-type Tar, for methylation of membrane-embedded receptors catalyzed by CheR (A and C) and demethylation catalyzed by phospho-CheB (B and D) in conditions as described previously (12) except CheR was 0.1 μM. Phospho-CheB was generated by the presence of an excess of phosphoramidate (12). (A and B) Rates for membrane-embedded receptors in the absence of attractant. (C and D) Altered rates upon occupancy with a saturating concentration (1 mM) of the Tar-linked attractant aspartate. Initial rates were determined by linear fits of time courses (12). Error bars are standard deviations.

FIG. 2.

Retention and elution of CheR and CheB in affinity columns carrying altered chemoreceptor pentapeptides. Cell extracts (E) containing methyltransferase (CheR) or methylesterase (CheB) were applied to affinity columns carrying the indicated peptide. Fractions were collected during application of the sample (F) in a column volume, with washing by three column volumes of buffer (B) and two column volumes of buffer containing the NWETF pentapeptide (P). Protein content of the same volume of each fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue. Representative results for the two families of altered peptides are shown in panels A and B, respectively.