An unorthodox sensory adaptation site in the E. coli serine chemoreceptor

The serine chemoreceptor of E. coli contains four canonical methylation sites for sensory adaptation that lie near inter-subunit helix interfaces of the Tsr homodimer. An unexplored fifth methylation site, E502, lies at an intra-subunit helix interface, closest to the HAMP domain that controls input-output signaling in methyl-accepting chemotaxis proteins. We analyzed, with in vivo FRET kinase assays, the serine thresholds and response cooperativities of Tsr receptors with different mutationally imposed modifications at sites 1-4 and/or at site 5. Tsr variants carrying E or Q at residue 502 in combination with unmodifiable D and N replacements at adaptation sites 1-4, underwent both methylation and demethylation/deamidation, although detection of the latter modifications required elevated intracellular levels of CheB. These Tsr variants could not mediate a chemotactic response to serine spatial gradients, demonstrating that adaptational modifications at E502 alone are not sufficient for Tsr function. Moreover, E502 is not critical for Tsr function because only two amino acid replacements at this residue abrogated serine chemotaxis: Tsr-E502P had extreme kinase-off output; Tsr-E502I had extreme kinase-on output. These large threshold shifts are probably due to the unique HAMP-proximal location of methylation site 5. However, a methylationmimicking glutamine at any Tsr modification site raised the serine response threshold, suggesting that all sites influence signaling by the same general mechanism, presumably through changes in packing stability of the methylation helix bundle. These findings are consistent with control of input-output signaling in Tsr through dynamic interplay of the structural stabilities of the HAMP and methylation bundles.