UNCOUPLED PHOSPHORYLATION AND ACTIVATION IN BACTERIAL CHEMOTAXIS - THE 2.3 ANGSTROM STRUCTURE OF AN ASPARTATE TO LYSINE MUTANT AT POSITION-13 OF CHEY

An aspartate to lysine mutation at position 13 of the chemotaxis regul atory protein CheY causes a constitutive tumbly phenotype when express ed at high copy number in vivo even though the mutant protein is not p hosphorylatable. These properties suggest that the D13K mutant adopts the active, signaling conformation of CheY independent of phosphorylat ion, so knowledge of its structure could explain the activation mechan ism of CheY. The x-ray crystallographic structure of the CheY D13K mut ant has been solved and refined at 2.3 Angstrom resolution to an R-fac tor of 14.3%. The mutant molecule shows no significant differences in backbone conformation when compared with the wild-type, Mg2+-free stru cture, but there are localized changes within the active site. The sid e chain of lysine 13 blocks access to the active site, whereas its eps ilon-amino group has no bonding interactions with other groups in the region. Also in the active site, the bond between lysine 109 and aspar tate 57 is weakened, and the solvent structure is perturbed. Although the D13K mutant has the inactive conformation in the crystalline form, rearrangements in the active site appear to weaken the overall struct ure of that region, potentially creating a metastable state of the mol ecule. If a conformational change is required for signaling by CheY D1 3K, then it most likely proceeds dynamically, in solution.