Crystal Structure of the CheA Histidine Phosphotransfer Domain that Mediates Response Regulator Phosphorylation in Bacterial Chemotaxis

Mourey, Lionel; Re, Sandra Da; Pédelacq, J.D.; Tolstykh, Tatiana; Faurie, Cécile; Guillet, Valérie; Stock, Jeffry B.; Samama, Jean‐Pierre

doi:10.1074/jbc.m101943200

Cited by 80 publications

(100 citation statements)

References 61 publications

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“…To correlate our structural model directly with the wealth of information available from mutational analysis and proteinprotein interaction studies in E. coli, homology models of corresponding components of the E. coli receptor array were constructed based on atomic structures of P3/P4/P5 (18) and P4/P5/ CheW (16) from T. maritima and P1 from Salmonella (19). We then built a large complex of P3/P4/P5 together with CheW, by aligning the three crystal structures (16,18,20) together (Materials and Methods).…”

Section: Resultsmentioning

confidence: 99%

“…Significant progress has been made in determining the atomic structures of the individual components of the core signaling complex, including the cytoplasmic domains from E. coli Tsr (8) and Thermotoga maritima MCP 1143C (16) and the various domains of CheA (17)(18)(19) and CheW (12,20). The interactions of some of the key components have been characterized by intensive studies using X-ray crystallography, NMR, ESR spectroscopy, chemical interaction mapping, and disulfide cross-linking (11,16,(21)(22)(23)(24).…”

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confidence: 99%

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Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells

Li¹,

Morado

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

187

339

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The chemoreceptors of Escherichia coli localize to the cell poles and form a highly ordered array in concert with the CheA kinase and the CheW coupling factor. However, a high-resolution structure of the array has been lacking, and the molecular basis of array assembly has thus remained elusive. Here, we use cryoelectron tomography of flagellated E. coli minicells to derive a 3D map of the intact array. Docking of high-resolution structures into the 3D map provides a model of the core signaling complex, in which a CheA/CheW dimer bridges two adjacent receptor trimers via multiple hydrophobic interactions. A further, hitherto unknown, hydrophobic interaction between CheW and the homologous P5 domain of CheA in an adjacent core complex connects the complexes into an extended array. This architecture provides a structural basis for array formation and could explain the high sensitivity and cooperativity of chemotaxis signaling in E. coli.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells

Li¹,

Morado

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

187

339

View full text Add to dashboard Cite

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“…Since A42 is fully solvent-exposed in the free domain and the 5FM-modified protein binds normally to the core complex ( Figure 3A), it is unlikely that modification of this position misfolds the protein. Nor is the native Ala side chain involved in the hydrogen bond network that controls the side chain conformation of H48 (12). Instead, the simplest explanation is that the additional bulk of Cys and 5FM modifications at the A42 position introduces a steric block into the substrate face of P1 essential for docking to the P4 catalytic domain during autophosphorylation.…”

Section: Internal Controlsmentioning

confidence: 99%

“…Yellow marks catalytic residues including H48, the site of phosphorylation on the P1 domain, and the ATP binding residues on the P4 domain. The composite CheA structure is assembled from S. typhimurium P1 domain (12), E. coli P2 domain (10), and T. maritima P3+P4+P5 domain fragment (11). Dashed lines indicate extended flexible linkers of undetermined structure that connect the domains.…”

Section: New Working Models For the Architecture Of The Core Complexmentioning

confidence: 99%

CheA Kinase of Bacterial Chemotaxis: Chemical Mapping of Four Essential Docking Sites

et al. 2006

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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-β) 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.The homodimeric, histidine kinase CheA is the central processing unit of the conserved signaling pathway that controls chemotaxis in Escherichia coli, Salmonella typhimurium, and many other bacteria (for reviews see refs 1-7). This pathway is the prototype for the large, ubiquitous class of two-component pathways that regulate a wide array of cell processes in prokaryotes. CheA and the other pathway components assemble to form large clusters of signaling complexes at the poles of the cell. The minimal signaling unit required for receptorregulated kinase activity, termed the core complex, comprises the oligomeri...

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“…There is a great deal of flexibility in the CheA structure, and we do not know how the P1 and P2 domains are arranged relative to P3, P4, and P5. There is, however, additional density in the reconstruction, and we can accommodate the structures of the P1 (22) and P2 (23) domains within this density.…”

Section: Location and Stoichiometry Of The Tar Cytoplasmic Domainmentioning

confidence: 99%

Three-dimensional structure and organization of a receptor/signaling complex

Francis

Wolanin

Stock

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Transmembrane signaling in bacterial chemotaxis has become an important model system for experimental and theoretical studies. These studies have provided a wealth of detailed molecular structures, including the structures of CheA, CheW, and the cytoplasmic domain of the serine receptor Tsr. How these three proteins interact to form the receptor͞signaling complex remains unknown. By using EM and single-particle image analysis, we present a three-dimensional reconstruction of the receptor͞signaling complex. The complex contains CheA, CheW, and the cytoplasmic portion of the aspartate receptor Tar. We observe density consistent with a structure containing 24 aspartate-receptor monomers and additional density sufficient to house the expected four CheA monomers and six CheW monomers. Within this bipolar structure are four groups of three receptor dimers that are not threefold symmetric and are therefore unlike the symmetric trimers observed in the x-ray crystal structure of the cytoplasmic domain of the serine receptor. In the latter, the interdimer contacts occur in the signaling domains near the hairpin loop. In our structure, the signaling domains within trimers appear spaced apart by the presence of CheA and CheW. This structure argues against models where one CheA and one CheW bind to the outer face of each of the dimers in the trimer. This structure of the receptor͞signaling complex provides an additional basis for understanding the architecture of the large arrays of chemotaxis receptors, CheA, and CheW found at the cell poles in motile bacteria.chemotaxis ͉ electron microscopy ͉ macromolecular assembly ͉ signal transduction M otile bacteria are capable of detecting and moving in response to gradients of nutrients, oxygen, light, and other stimuli. They do so in response to temporal changes in the stimuli by altering the behavior of the motors that generate the force for cell movement. The signal transduction system controlling bacterial chemotaxis has become an important model for the study of transmembrane signaling, and the most studied system is that in Escherichia coli.The core components of the E. coli chemotaxis signaltransduction system are found in nearly all motile bacteria and consist of transmembrane chemoreceptors; an associated histidine kinase, CheA; and an associated CheA-activator, CheW. Signaling occurs within the context of the assembled receptor͞ CheW͞CheA complexes within a large, densely packed patch of chemoreceptors, which is typically located at one (or both) poles of the cell (1). No gross structural changes in the receptor͞ signaling complex are observed in response to stimulatory ligands (2).Recent studies and modeling of the chemotaxis system have emphasized the importance of interactions among large numbers of receptors in terms of the system's sensitivity and adaptive response (3, 4), and chemoreceptors for different ligands appear to communicate with one another (3). The lack of detailed information about how CheA and CheW interact with the chemoreceptors and each other in the ...

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Crystal Structure of the CheA Histidine Phosphotransfer Domain that Mediates Response Regulator Phosphorylation in Bacterial Chemotaxis

Cited by 80 publications

References 61 publications

Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells

Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells

CheA Kinase of Bacterial Chemotaxis: Chemical Mapping of Four Essential Docking Sites

Three-dimensional structure and organization of a receptor/signaling complex

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