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Protein footprinting in a complex milieu: identifying the interaction surfaces of the chemotaxis adaptor protein CheW.

ABSTRACT: 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.

SUBMITTER: Underbakke ES

PROVIDER: S-EPMC3179904 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Protein footprinting in a complex milieu: identifying the interaction surfaces of the chemotaxis adaptor protein CheW.

Underbakke Eric S ES Zhu Yimin Y Kiessling Laura L LL

Journal of molecular biology 20110402 4

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 base ...[more]

PMID: 21463637

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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 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:RNA-binding proteins (RBPs) are intimately involved in all aspects of RNA processing and regulation and are linked to neurodegenerative diseases and cancer. Therefore, understanding the relationship between RBPs and their RNA targets is critical for a broader understanding of post-transcriptional regulation in normal and disease processes. The majority of approaches to study RNA-protein interactions focus on single RBPs, however there are many hundreds RBPs encoded in the human genome, and each cell type expresses a different catalog of these regulatory molecules, greatly limiting the ability of these single RBP approaches to capture the global landscape of RNA-protein interactions. We and others have developed approaches to globally catalog regions of mRNAs that are bound by proteins in an unbiased manner. Here we describe a detailed protocol for performing our RNase-mediated protein footprint sequencing approach, termed protein interaction profile sequencing (PIP-seq). In this protocol, RNA-protein interactions are stabilized by cross-linking, and un-bound regions are digested with RNases, leaving only the protein-bound regions intact. To control for RNase insensitive regions, proteins are first denatured and then RNP complexes are subject to RNase treatment. After high-throughput sequencing of remaining fragments, peak calling is performed to identify protein protected sites (PPSs). We describe the application of this protocol to a human embryonic kidney cell line and perform basic quality control, reproducibility and benchmarking analyses. Finally, we describe the landscape of protein-interactions in HEK293T cells, underscoring the value of this approach. Future applications of this method to study the dynamics of RNA-protein interactions in developmental processes will help to uncover the role of RBPs in post-transcriptional regulation. Protein interaction profile sequencing (PIP-seq) in HEK293T cells. Three replicates of formaldehyde cross-linked PIP-seq are described

Dataset Information

Protein footprinting in a complex milieu: identifying the interaction surfaces of the chemotaxis adaptor protein CheW.

Publications

Protein footprinting in a complex milieu: identifying the interaction surfaces of the chemotaxis adaptor protein CheW.

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