Dataset Information

The 3.2 A resolution structure of a receptor: CheA:CheW signaling complex defines overlapping binding sites and key residue interactions within bacterial chemosensory arrays.

ABSTRACT: 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.

SUBMITTER: Li X

PROVIDER: S-EPMC3694592 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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The 3.2 Å resolution structure of a receptor: CheA:CheW signaling complex defines overlapping binding sites and key residue interactions within bacterial chemosensory arrays.

Li Xiaoxiao X Fleetwood Aaron D AD Bayas Camille C Bilwes Alexandrine M AM Ortega Davi R DR Falke Joseph J JJ Zhulin Igor B IB Crane Brian R BR

Biochemistry 20130523 22

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 ...[more]

PMID: 23668907

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Project description:Salmonella enterica is the most frequently reported cause of foodborne illness. As in other microorganisms, chemotaxis affords key physiological benefits, including enhanced access to growth substrates, but also plays an important role in infection and disease. Chemoreceptor signaling core complexes, consisting of CheA, CheW and methyl-accepting chemotaxis proteins (MCPs), modulate the switching of bacterial flagella rotation that drives cell motility. These complexes, through the formation of heterohexameric rings composed of CheA and CheW, form large clusters at the cell poles. RecA plays a key role in polar cluster formation, impairing the assembly when the SOS response is activated. In this study, we determined that RecA protein interacts with both CheW and CheA. The binding of these proteins to RecA is needed for wild-type polar cluster formation. In silico models showed that one RecA molecule, attached to one signaling unit, fits within a CheA-CheW ring without interfering with the complex formation or array assembly. Activation of the SOS response is followed by an increase in RecA, which rises up the number of signaling complexes associated with this protein. This suggests the presence of allosteric inhibition in the CheA-CheW interaction and thus of heterohexameric ring formation, impairing the array assembly. STED imaging demonstrated that all core unit components (CheA, CheW, and MPCs) have the same subcellular location as RecA. Activation of the SOS response promotes the RecA distribution along the cell instead of being at the cell poles. CheA- and CheW- RecA interactions are also crucial for chemotaxis, which is maintained when the SOS response is induced and the signaling units are dispersed. Our results provide new molecular-level insights into the function of RecA in chemoreceptor clustering and chemotaxis determining that the impaired chemoreceptor clustering not only inhibits swarming but also modulates chemotaxis in SOS-induced cells, thereby modifying bacterial motility in the presence of DNA-damaging compounds, such as antibiotics.

Project description:The ultrasensitive, ultrastable bacterial chemosensory array of Escherichia coli and Salmonella typhimurium is representative of the large, conserved family of sensory arrays that control the cellular chemotaxis of motile bacteria and Archaea. The core framework of the membrane-bound array is a lattice assembled from three components: a transmembrane receptor, a cytoplasmic His kinase (CheA), and a cytoplasmic adaptor protein (CheW). Structural studies in the field have revealed the global architecture of the array and complexes between specific components, but much remains to be learned about the essential protein-protein interfaces that define array structure and transmit signals between components. This study has focused on the structure, function, and on-off switching of a key contact between the kinase and adaptor proteins in the working, membrane-bound array. Specifically, the study addressed interface 1 in the putative kinase-adaptor ring where subdomain 1 of the kinase regulatory domain contacts subdomain 2 of the adaptor protein. Two independent approaches, disulfide mapping and site-directed Trp and Ala mutagenesis, were employed (i) to test the structural model of interface 1 and (ii) to investigate its functional roles in both stable kinase incorporation and receptor-regulated kinase on-off switching. Studies were conducted in functional, membrane-bound arrays or in live cells. The findings reveal that crystal structures of binary and ternary complexes accurately depict the native interface in its kinase-activating on state. Furthermore, the findings indicate that at least part of the interface becomes less closely packed in its kinase-inhibiting off state. Together, the evidence shows the interface has a dual structural and signaling function that is crucial for incorporation of the stable kinase into the array, for kinase activation in the array on state, and likely for attractant-triggered kinase on-off switching. A model is presented that describes the concerted transmission of a conformational signal among the receptor, the kinase regulatory domain, and the adaptor protein. In principle, this signal could spread out into the surrounding array via the kinase-adaptor ring, employing a series of alternating frozen-dynamic transitions that transmit low-energy attractant signals long distances.

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The 3.2 A resolution structure of a receptor: CheA:CheW signaling complex defines overlapping binding sites and key residue interactions within bacterial chemosensory arrays.

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The 3.2 Å resolution structure of a receptor: CheA:CheW signaling complex defines overlapping binding sites and key residue interactions within bacterial chemosensory arrays.

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