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Regulation of the chemotaxis histidine kinase CheA: A structural perspective.

ABSTRACT: Bacteria sense and respond to their environment through a highly conserved assembly of transmembrane chemoreceptors (MCPs), the histidine kinase CheA, and the coupling protein CheW, hereafter termed "the chemosensory array". In recent years, great strides have been made in understanding the architecture of the chemosensory array and how this assembly engenders sensitive and cooperative responses. Nonetheless, a central outstanding question surrounds how receptors modulate the activity of the CheA kinase, the enzymatic output of the sensory system. With a focus on recent advances, we summarize the current understanding of array structure and function to comment on the molecular mechanism by which CheA, receptors and CheW generate the high sensitivity, gain and dynamic range emblematic of bacterial chemotaxis. The complexity of the chemosensory arrays has motivated investigation with many different approaches. In particular, structural methods, genetics, cellular activity assays, nanodisc technology and cryo-electron tomography have provided advances that bridge length scales and connect molecular mechanism to cellular function. Given the high degree of component integration in the chemosensory arrays, we ultimately aim to understand how such networked molecular interactions generate a whole that is truly greater than the sum of its parts. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

SUBMITTER: Muok AR

PROVIDER: S-EPMC7212787 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Regulation of the chemotaxis histidine kinase CheA: A structural perspective.

Muok Alise R AR Briegel Ariane A Crane Brian R BR

Biochimica et biophysica acta. Biomembranes 20190730 1

Bacteria sense and respond to their environment through a highly conserved assembly of transmembrane chemoreceptors (MCPs), the histidine kinase CheA, and the coupling protein CheW, hereafter termed "the chemosensory array". In recent years, great strides have been made in understanding the architecture of the chemosensory array and how this assembly engenders sensitive and cooperative responses. Nonetheless, a central outstanding question surrounds how receptors modulate the activity of the Che ...[more]

PMID: 31374212

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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.

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.

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Regulation of the chemotaxis histidine kinase CheA: A structural perspective.

Publications

Regulation of the chemotaxis histidine kinase CheA: A structural perspective.

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