Dataset Information

A bifunctional kinase-phosphatase in bacterial chemotaxis.

ABSTRACT: Phosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coli chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate that an unusual CheA homologue, CheA(3), is not only a phosphodonor for the principal CheY protein, CheY(6), but is also is a specific phosphatase for CheY(6)-P. This phosphatase activity accelerates CheY(6)-P dephosphorylation to a rate that is comparable with the measured stimulus response time of approximately 1 s. CheA(3) possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (P5) domains, which are joined by a 794-amino acid sequence that is required for phosphatase activity. The P1 domain of CheA(3) is phosphorylated by CheA(4), and it subsequently acts as a phosphodonor for the response regulators. A CheA(3) mutant protein without the 794-amino acid region lacked phosphatase activity, retained phosphotransfer function, but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach, we showed that a 200-amino acid segment of CheA(3) is required for phosphatase activity. The phosphatase activity of previously identified nonhybrid histidine protein kinases depends on the dimerization and histidine phosphorylation (DHp) domains. However, CheA(3) lacks a DHp domain, suggesting that its phosphatase mechanism is different from that of other histidine protein kinases.

SUBMITTER: Porter SL

PROVIDER: S-EPMC2587623 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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A bifunctional kinase-phosphatase in bacterial chemotaxis.

Porter Steven L SL Roberts Mark A J MA Manning Cerys S CS Armitage Judith P JP

Proceedings of the National Academy of Sciences of the United States of America 20081119 47

Phosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coli chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate th ...[more]

PMID: 19020080

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Project description:The chemotaxis pathway of Escherichia coli and Salmonella typhimurium is the paradigm for the ubiquitous class of 2-component signaling pathways in prokaryotic organisms. Chemosensing begins with the binding of a chemical attractant to a transmembrane receptor on the cell surface. The resulting transmembrane signal regulates a cytoplasmic, multiprotein signaling complex that controls cellular swimming behavior by generating a diffusible phosphoprotein. The minimal functional unit of this signaling complex, termed the core complex, consists of the transmembrane receptor, the coupling protein CheW, and the histidine kinase CheA. Though the structures of individual components are largely known and the core complex can be functionally reconstituted, the architecture of the assembled core complex has remained elusive. To probe this architecture, the present study has utilized an enhanced version of the protein-interactions-by-cysteine-modification method (PICM-beta) to map out docking surfaces on CheA essential for kinase activity and for core complex assembly. The approach employed a library of 70 single, engineered cysteine residues, scattered uniformly over the surfaces of the five CheA domains in a cysteine-free CheA background. These surface Cys residues were further modified by the sulfhydryl-specific alkylating agent, 5-fluorescein-maleimide (5FM). The functional effects of individual Cys and 5FM-Cys surface modifications were measured by kinase assays of CheA activity in both the free and core complex-associated states, and by direct binding assays of CheA associations with CheW and the receptor. The results define (i) two mutual docking surfaces on the CheA substrate and catalytic domains essential for the association of these domains during autophosphorylation, (ii) a docking surface on the CheA regulatory domain essential for CheW binding, and (iii) a large docking surface encompassing regions of the CheA dimerization, catalytic, and regulatory domains proposed to bind the receptor. To test the generality of these findings, a CheA sequence alignment was analyzed, revealing that the newly identified docking surfaces are highly conserved among CheA homologues. These results strongly suggest that the same docking sites are widely utilized in prokaryotic sensory pathways. Finally, the results provide new structural constraints allowing the development of improved models for core complex architecture.

Project description:The histidine kinase CheA plays a central role in signal integration, conversion, and amplification in the bacterial chemotaxis signal transduction pathway. The kinase activity is regulated in chemotaxis signaling complexes formed via the interactions among CheA's regulatory domain (P5), the coupling protein CheW, and transmembrane chemoreceptors. Despite recent advancements in the understanding of the architecture of the signaling complex, the molecular mechanism underlying this regulation remains elusive. An interdomain linker that connects the catalytic (P4) and regulatory domains of CheA may mediate regulatory signals from the P5-CheW-receptor interactions to the catalytic domain. To investigate whether this interdomain linker is capable of both activating and inhibiting CheA, we performed in vivo screens to search for P4-P5 linker mutations that result in different CheA autokinase activities. Several CheA variants were identified with kinase activities ranging from 30% to 670% of the activity of wild-type CheA. All of these CheA variants were defective in receptor-mediated kinase activation, indicating that the natural receptor-mediated signal transmission pathway was simultaneously affected by these mutations. The altered P4-P5 linkers were sufficient for making significant changes in the kinase activity even in the absence of the P5 domain. Therefore, the interdomain linker is an active module that has the ability to impose regulatory effects on the catalytic activity of the P4 domain. These results suggest that chemoreceptors may manipulate the conformation of the P4-P5 linker to achieve CheA regulation in the platform of the signaling complex.IMPORTANCE The molecular mechanism underlying kinase regulation in bacterial chemotaxis signaling complexes formed by the regulatory domain of the histidine kinase CheA, the coupling protein CheW, and chemoreceptors is still unknown. We isolated and characterized mutations in the interdomain linker that connects the catalytic and regulatory domains of CheA and found that the linker mutations resulted in different CheA autokinase activities in the absence and presence of the regulatory domain as well as a defect in receptor-mediated kinase activation. These results demonstrate that the interdomain linker is an active module that has the ability to impose regulatory effects on CheA activity. Chemoreceptors may manipulate the conformation of this interdomain linker to achieve CheA regulation in the platform of the signaling complex.

Dataset Information

A bifunctional kinase-phosphatase in bacterial chemotaxis.

Publications

A bifunctional kinase-phosphatase in bacterial chemotaxis.

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