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Crystal structures of beryllium fluoride-free and beryllium fluoride-bound CheY in complex with the conserved C-terminal peptide of CheZ reveal dual binding modes specific to CheY conformation.

ABSTRACT: Chemotaxis, the environment-specific swimming behavior of a bacterial cell is controlled by flagellar rotation. The steady-state level of the phosphorylated or activated form of the response regulator CheY dictates the direction of flagellar rotation. CheY phosphorylation is regulated by a fine equilibrium of three phosphotransfer activities: phosphorylation by the kinase CheA, its auto-dephosphorylation and dephosphorylation by its phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two spatially distinct protein-protein contacts: tethering of the two proteins to each other and formation of an active site for dephosphorylation. The former involves interaction of phosphorylated CheY with the small highly conserved C-terminal helix of CheZ (CheZ(C)), an indispensable structural component of the functional CheZ protein. To understand how the CheZ(C) helix, representing less than 10% of the full-length protein, ascertains molecular specificity of binding to CheY, we have determined crystal structures of CheY in complex with a synthetic peptide corresponding to 15 C-terminal residues of CheZ (CheZ(200-214)) at resolutions ranging from 2.0 A to 2.3A. These structures provide a detailed view of the CheZ(C) peptide interaction both in the presence and absence of the phosphoryl analog, BeF3-. Our studies reveal that two different modes of binding the CheZ(200-214) peptide are dictated by the conformational state of CheY in the complex. Our structures suggest that the CheZ(C) helix binds to a "meta-active" conformation of inactive CheY and it does so in an orientation that is distinct from the one in which it binds activated CheY. Our dual binding mode hypothesis provides implications for reverse information flow in CheY and extends previous observations on inherent resilience in CheY-like signaling domains.

SUBMITTER: Guhaniyogi J

PROVIDER: S-EPMC3666561 | biostudies-literature | 2006 Jun

REPOSITORIES: biostudies-literature

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Crystal structures of beryllium fluoride-free and beryllium fluoride-bound CheY in complex with the conserved C-terminal peptide of CheZ reveal dual binding modes specific to CheY conformation.

Guhaniyogi Jayita J Robinson Victoria L VL Stock Ann M AM

Journal of molecular biology 20060406 3

Chemotaxis, the environment-specific swimming behavior of a bacterial cell is controlled by flagellar rotation. The steady-state level of the phosphorylated or activated form of the response regulator CheY dictates the direction of flagellar rotation. CheY phosphorylation is regulated by a fine equilibrium of three phosphotransfer activities: phosphorylation by the kinase CheA, its auto-dephosphorylation and dephosphorylation by its phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ r ...[more]

PMID: 16674976

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Project description:Microorganisms and plants utilize two-component systems to regulate adaptive responses to changing environmental conditions. Sensor kinases detect stimuli and alter their autophosphorylation activity accordingly. Signal propagation occurs via the transfer of phosphoryl groups from upstream kinases to downstream response regulator proteins. Removal of phosphoryl groups from the response regulator typically resets the system. Members of the same protein family may catalyze phosphorylation and dephosphorylation reactions with different efficiencies, exhibiting rate constants spanning many orders of magnitude to accommodate response time scales from milliseconds to days. We previously found that variable positions one or two residues to the C-terminal side of the conserved Asp phosphorylation site (D+2) or Thr/Ser (T+1/T+2) in response regulators alter reaction kinetics by direct interaction with phosphodonor or phosphoacceptor molecules. Here, we explore the kinetic effects of amino acid substitutions at the two positions immediately C-terminal to the conserved Lys (K+1/K+2) in the model Escherichia coli response regulator CheY. We measured CheY autophosphorylation and autodephosphorylation rate constants for 27 pairs of K+1/K+2 residues that represent 84% of naturally occurring response regulators. Effects on autodephosphorylation were modest, but autophosphorylation rate constants varied by 2 orders of magnitude, suggesting that the K+1/K+2 positions influence reaction kinetics by altering the conformational spectrum sampled by CheY at equilibrium. Additional evidence supporting this indirect mechanism includes the following: the effect on autophosphorylation rate constants is independent of the phosphodonor, the autophosphorylation rate constants and dissociation constants for the phosphoryl group analog BeF3 - are inversely correlated, and the K+1/K+2 positions are distant from the phosphorylation site.IMPORTANCE We have identified five variable positions in response regulators that allow the rate constants of autophosphorylation and autodephosporylation reactions each to be altered over 3 orders of magnitude in CheY. The distributions of variable residue combinations across response regulator subfamilies suggest that distinct mechanisms associated with different variable positions allow reaction rates to be tuned independently during evolution for diverse biological purposes. This knowledge could be used in synthetic-biology applications to adjust the properties (e.g., background noise and response duration) of biosensors and may allow prediction of response regulator reaction kinetics from the primary amino acid sequence.

Dataset Information

Crystal structures of beryllium fluoride-free and beryllium fluoride-bound CheY in complex with the conserved C-terminal peptide of CheZ reveal dual binding modes specific to CheY conformation.

Publications

Crystal structures of beryllium fluoride-free and beryllium fluoride-bound CheY in complex with the conserved C-terminal peptide of CheZ reveal dual binding modes specific to CheY conformation.

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