Noncritical Signaling Role of a Kinase-Receptor Interaction Surface in the Escherichia coli Chemosensory Core Complex.
ABSTRACT: In Escherichia coli chemosensory arrays, transmembrane receptors, a histidine autokinase CheA, and a scaffolding protein CheW interact to form an extended hexagonal lattice of signaling complexes. One interaction, previously assigned a crucial signaling role, occurs between chemoreceptors and the CheW-binding P5 domain of CheA. Structural studies showed a receptor helix fitting into a hydrophobic cleft at the boundary between P5 subdomains. Our work aimed to elucidate the in vivo roles of the receptor-P5 interface, employing as a model the interaction between E. coli CheA and Tsr, the serine chemoreceptor. Crosslinking assays confirmed P5 and Tsr contacts in vivo and their strict dependence on CheW. Moreover, the P5 domain only mediated CheA recruitment to polar receptor clusters if CheW was also present. Amino acid replacements at CheA.P5 cleft residues reduced CheA kinase activity, lowered serine response cooperativity, and partially impaired chemotaxis. Pseudoreversion studies identified suppressors of P5 cleft defects at other P5 groove residues or at surface-exposed residues in P5 subdomain 1, which interacts with CheW in signaling complexes. Our results indicate that a high-affinity P5-receptor binding interaction is not essential for core complex function. Rather, P5 groove residues are probably required for proper cleft structure and/or dynamic behavior, which likely impact conformational communication between P5 subdomains and the strong binding interaction with CheW that is necessary for kinase activation. We propose a model for signal transmission in chemotaxis signaling complexes in which the CheW-receptor interface plays the key role in conveying signaling-related conformational changes from receptors to the CheA kinase.
Project description:Bacterial chemosensory arrays are composed of extended networks of chemoreceptors (also known as methyl-accepting chemotaxis proteins, MCPs), the histidine kinase CheA, and the adaptor protein CheW. Models of these arrays have been developed from cryoelectron microscopy, crystal structures of binary and ternary complexes, NMR spectroscopy, mutational, data and biochemical studies. A new 3.2 Å resolution crystal structure of a Thermotoga maritima MCP protein interaction region in complex with the CheA kinase-regulatory module (P4-P5) and adaptor protein CheW provides sufficient detail to define residue contacts at the interfaces formed among the three proteins. As in a previous 4.5 Å resolution structure, CheA-P5 and CheW interact through conserved hydrophobic surfaces at the ends of their ?-barrels to form pseudo 6-fold symmetric rings in which the two proteins alternate around the circumference. The interface between P5 subdomain 1 and CheW subdomain 2 was anticipated from previous studies, whereas the related interface between CheW subdomain 1 and P5 subdomain 2 has only been observed in these ring assemblies. The receptor forms an unexpected structure in that the helical hairpin tip of each subunit has "unzipped" into a continuous ?-helix; four such helices associate into a bundle, and the tetramers bridge adjacent P5-CheW rings in the lattice through interactions with both P5 and CheW. P5 and CheW each bind a receptor helix with a groove of conserved hydrophobic residues between subdomains 1 and 2. P5 binds the receptor helix N-terminal to the tip region (lower site), whereas CheW binds the same helix with inverted polarity near the bundle end (upper site). Sequence comparisons among different evolutionary classes of chemotaxis proteins show that the binding partners undergo correlated changes at key residue positions that involve the lower site. Such evolutionary analyses argue that both CheW and P5 bind to the receptor tip at overlapping positions. Computational genomics further reveal that two distinct CheW proteins in Thermotogae utilize the analogous recognition motifs to couple different receptor classes to the same CheA kinase. Important residues for function previously identified by mutagenesis, chemical modification and biophysical approaches also map to these same interfaces. Thus, although the native CheW-receptor interaction is not observed in the present crystal structure, the bioinformatics and previous data predict key features of this interface. The companion study of the P5-receptor interface in native arrays (accompanying paper Piasta et al. (2013) Biochemistry, DOI: 10.1021/bi400385c) shows that, despite the non-native receptor fold in the present crystal structure, the local helix-in-groove contacts of the crystallographic P5-receptor interaction are present in native arrays and are essential for receptor regulation of kinase activity.
Project description:The signaling apparatus that controls bacterial chemotaxis is composed of a core complex containing chemoreceptors, the histidine autokinase CheA, and the coupling protein CheW. Site-specific spin labeling and pulsed dipolar ESR spectroscopy (PDS) have been applied to investigate the structure of a soluble ternary complex formed by Thermotoga maritima CheA (TmCheA), CheW, and receptor signaling domains. Thirty-five symmetric spin-label sites (SLSs) were engineered into the five domains of the CheA dimer and CheW to provide distance restraints within the CheA:CheW complex in the absence and presence of a soluble receptor that inhibits kinase activity (Tm14). Additional PDS restraints among spin-labeled CheA, CheW, and an engineered single-chain receptor labeled at six different sites allow docking of the receptor structure relative to the CheA:CheW complex. Disulfide cross-linking between selectively incorporated Cys residues finds two pairs of positions that provide further constraints within the ternary complex: one involving Tm14 and CheW and another involving Tm14 and CheA. The derived structure of the ternary complex indicates a primary site of interaction between CheW and Tm14 that agrees well with previous biochemical and genetic data for transmembrane chemoreceptors. The PDS distance distributions are most consistent with only one CheW directly engaging one dimeric Tm14. The CheA dimerization domain (P3) aligns roughly antiparallel to the receptor-conserved signaling tip but does not interact strongly with it. The angle of the receptor axis with respect to P3 and the CheW-binding P5 domains is bound by two limits differing by approximately 20 degrees . In one limit, Tm14 aligns roughly along P3 and may interact to some extent with the hinge region near the P3 hairpin loop. In the other limit, Tm14 tilts to interact with the P5 domain of the opposite subunit in an interface that mimics that observed with the P5 homologue CheW. The time domain ESR data can be simulated from the model only if orientational variability is introduced for the P5 and, especially, P3 domains. The Tm14 tip also binds beside one of the CheA kinase domains (P4); however, in both bound and unbound states, P4 samples a broad range of distributions that are only minimally affected by Tm14 binding. The CheA P1 domains that contain the substrate histidine are also broadly distributed in space under all conditions. In the context of the hexagonal lattice formed by trimeric transmembrane chemoreceptors, the PDS structure is best accommodated with the P3 domain in the center of a honeycomb edge.
Project description:Characterizing protein-protein interactions in a biologically relevant context is important for understanding the mechanisms of signal transduction. Most signal transduction systems are membrane associated and consist of large multiprotein complexes that undergo rapid reorganization--circumstances that present challenges to traditional structure determination methods. To study protein-protein interactions in a biologically relevant complex milieu, we employed a protein footprinting strategy based on isotope-coded affinity tag (ICAT) reagents. ICAT reagents are valuable tools for proteomics. Here, we show their utility in an alternative application--they are ideal for protein footprinting in complex backgrounds because the affinity tag moiety allows for enrichment of alkylated species prior to analysis. We employed a water-soluble ICAT reagent to monitor cysteine accessibility and thereby to identify residues involved in two different protein-protein interactions in the Escherichia coli chemotaxis signaling system. The chemotaxis system is an archetypal transmembrane signaling pathway in which a complex protein superstructure underlies sophisticated sensory performance. The formation of this superstructure depends on the adaptor protein CheW, which mediates a functionally important bridging interaction between transmembrane receptors and histidine kinase. ICAT footprinting was used to map the surfaces of CheW that interact with the large multidomain histidine kinase CheA, as well as with the transmembrane chemoreceptor Tsr in native E. coli membranes. By leveraging the affinity tag, we successfully identified CheW surfaces responsible for CheA-Tsr interaction. The proximity of the CheA and Tsr binding sites on CheW suggests the formation of a composite CheW-Tsr surface for the recruitment of the signaling kinase to the chemoreceptor complex.
Project description:The Escherichia coli chemosensory system consists of large arrays of transmembrane chemoreceptors associated with a dedicated histidine kinase, CheA, and a linker protein, CheW, that couples CheA activity to receptor control. The kinase activity responses to receptor ligand occupancy changes can be highly cooperative, reflecting allosteric coupling of multiple CheA and receptor molecules. Recent structural and functional studies have led to a working model in which receptor core complexes, the minimal units of signaling, are linked into hexagonal arrays through a unique interface 2 interaction between CheW and the P5 domain of CheA. To test this array model, we constructed and characterized CheA and CheW mutants with amino acid replacements at key interface 2 residues. The mutant proteins proved defective in interface 2-specific in vivo cross-linking assays, and formed signaling complexes that were dispersed around the cell membrane rather than clustered at the cell poles as in wild type chemosensory arrays. Interface 2 mutants down-regulated CheA activity in response to attractant stimuli in vivo, but with much less cooperativity than the wild type. Moreover, mutant cells containing fluorophore-tagged receptors exhibited greater basal anisotropy that changed rapidly in response to attractant stimuli, consistent with facile changes in loosely packed receptors. We conclude that interface 2 lesions disrupt important network connections between core complexes, preventing receptors from operating in large, allosteric teams. This work confirms the critical role of interface 2 in organizing the chemosensory array, in directing the clustered array to the cell poles, and in producing its highly cooperative signaling properties.
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 <i>in vivo</i> 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.<b>IMPORTANCE</b> 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: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 receptor/signaling complex remains unknown. By using EM and single-particle image analysis, we present a three-dimensional reconstruction of the receptor/signaling 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 receptor/signaling 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.
Project description: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.
Project description:Chemical signals sensed on the periplasmic side of bacterial cells by transmembrane chemoreceptors are transmitted to the flagellar motors via the histidine kinase CheA, which controls the phosphorylation level of the effector protein CheY. Chemoreceptor arrays comprise remarkably stable supramolecular structures in which thousands of chemoreceptors are networked through interactions between their cytoplasmic tips, CheA, and the small coupling protein CheW. To explore the conformational changes that occur within this protein assembly during signalling, we used in vivo cross-linking methods to detect close interactions between the coupling protein CheW and the serine receptor Tsr in intact Escherichia coli cells. We identified two signal-sensitive contacts between CheW and the cytoplasmic tip of Tsr. Our results suggest that ligand binding triggers changes in the receptor that alter its signalling contacts with CheW (and/or CheA).
Project description:Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors.
Project description:In bacterial chemotaxis, transmembrane chemoreceptors, the CheA histidine kinase, and the CheW coupling protein assemble into signaling complexes that allow bacteria to modulate their swimming behavior in response to environmental stimuli. Among the protein-protein interactions in the ternary complex, CheA-CheW and CheW-receptor interactions were studied previously, whereas CheA-receptor interaction has been less investigated. Here, we characterize the CheA-receptor interaction in Thermotoga maritima by NMR spectroscopy and validate the identified receptor binding site of CheA in Escherichia coli chemotaxis. We find that CheA interacts with a chemoreceptor in a manner similar to that of CheW, and the receptor binding site of CheA's regulatory domain is homologous to that of CheW. Collectively, the receptor binding sites in the CheA-CheW complex suggest that conformational changes in CheA are required for assembly of the CheA-CheW-receptor ternary complex and CheA activation.