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Volume 272, Number 18, Issue of May 2, 1997 pp. 11850-11855
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Uncoupled Phosphorylation and Activation in Bacterial Chemotaxis
THE 2.3 Å STRUCTURE OF AN ASPARTATE TO LYSINE MUTANT AT POSITION 13 OF CheY

(Received for publication, December 18, 1996)

Meiying Jiang , Robert B. Bourret ^¶ , Melvin I. Simon ^¶ and Karl Volz

From the  Department of Microbiology and Immunology, University of Illinois, Chicago, Illinois 60612 and the ^¶ Division of Biology, California Institute of Technology, Pasadena, California 91125

An aspartate to lysine mutation at position 13 of the chemotaxis regulatory protein CheY causes a constitutive tumbly phenotype when expressed at high copy number in vivo even though the mutant protein is not phosphorylatable. These properties suggest that the D13K mutant adopts the active, signaling conformation of CheY independent of phosphorylation, so knowledge of its structure could explain the activation mechanism of CheY. The x-ray crystallographic structure of the CheY D13K mutant has been solved and refined at 2.3 Å resolution to an R-factor of 14.3%. The mutant molecule shows no significant differences in backbone conformation when compared with the wild-type, Mg²⁺-free structure, but there are localized changes within the active site. The side chain of lysine 13 blocks access to the active site, whereas its -amino group has no bonding interactions with other groups in the region. Also in the active site, the bond between lysine 109 and aspartate 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 structure of that region, potentially creating a metastable state of the molecule. If a conformational change is required for signaling by CheY D13K, then it most likely proceeds dynamically, in solution.

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