Dataset Information

The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides.

ABSTRACT: CheY is a response regulator in bacterial chemotaxis. Escherichia coli CheY mutants T87I and T87I/Y106W CheY are phosphorylatable on Asp57 but unable to generate clockwise rotation of the flagella. To understand this phenotype in terms of structure, stable analogs of the two CheY-P mutants were synthesized: T87I phosphono-CheY and T87I phosphono-CheY. Dissociation constants for peptides derived from flagellar motor protein FliM and phosphatase CheZ were determined for phosphono-CheY and the two mutants. The peptides bind phosphono-CheY almost as strongly as CheY-P; however, they do not bind T87I phosphono-CheY or T87I/Y106W phosphono-CheY, implying that the mutant proteins cannot bind FliM or CheZ tightly in vivo. The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY were solved to resolutions of 1.8 and 2.4A, respectively. The increased bulk of I87 forces the side-chain of Y106 or W106, into a more solvent-accessible conformation, which occludes the peptide-binding site.

SUBMITTER: McAdams K

PROVIDER: S-EPMC2850201 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides.

McAdams Kenneth K Casper Eric S ES Matthew Haas R R Santarsiero Bernard D BD Eggler Aimee L AL Mesecar Andrew A Halkides Christopher J CJ

Archives of biochemistry and biophysics 20080905 2

CheY is a response regulator in bacterial chemotaxis. Escherichia coli CheY mutants T87I and T87I/Y106W CheY are phosphorylatable on Asp57 but unable to generate clockwise rotation of the flagella. To understand this phenotype in terms of structure, stable analogs of the two CheY-P mutants were synthesized: T87I phosphono-CheY and T87I phosphono-CheY. Dissociation constants for peptides derived from flagellar motor protein FliM and phosphatase CheZ were determined for phosphono-CheY and the two ...[more]

PMID: 18801331

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Project description:Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY ⋅ FliMN with and without D13K-Y106W have shown FliMN-bound CheY contains features of both active and inactive states. We used molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliMN and D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D57 phosphorylation site and the FliMN interface, namely the closure of the α4-β4 hinge and inward rotation of Y- or W106 with W58. We used hydroxy-radical foot printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other side chains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay side chains reported by XFMS confirmed the intermediate conformation of the native CheY ⋅ FliMN complex, the inactive state of free D13K-Y106W CheY, and the MD-based network architecture. We extended the MD analysis to determine temporal coupling and energetics during activation. Coupled aromatic residue rotation was a graded rather than a binary switch, with Y- or W106 side-chain burial correlated with increased FliMN affinity. Activation entrained CheY fold stabilization to FliMN affinity. The CheY network could be partitioned into four dynamically coordinated sectors. Residue substitutions mapped to sectors around D57 or the FliMN interface according to phenotype. FliMN increased sector size and interactions. These sectors fused between the substituted K13-W106 residues to organize a tightly packed core and novel surfaces that may bind additional sites to explain the cooperative motor response. The community maps provide a more complete description of CheY priming than proposed thus far.

Dataset Information

The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides.

Publications

The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides.

Similar Datasets

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