Identification of a Chemoreceptor for Tricarboxylic Acid Cycle Intermediates

Lacal, Jesús; Alfonso, Carlos; Liu, Xianxian; Parales, Rebecca E.; Morel, Bertrand; Conejero‐Lara, Francisco; Rivas, Germán; Duque, Estrella; Ramos, Juan L.; Krell, Tino

doi:10.1074/jbc.m110.110403

Cited by 87 publications

(103 citation statements)

References 57 publications

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“…In this alignment, the malate/succinate-binding pocket (proximal module) coincides with the acetate-binding pocket (distal module). McpS crystallized as a dimer, which is consistent with ultracentrifugation studies that demonstrated the existence of stable dimers in the presence of bound ligand (12). The dimer interface involves interactions between α1 and α4 of both monomers.…”

Section: Resultssupporting

confidence: 66%

“…The five oxygen atoms of malate are involved in eight hydrogen bonds with these five amino acids. The fact that malate is bound by amino acids from both monomers of the dimer provides an explanation for our previous observation that McpS-LBR monomers are unable to bind malate and that malate binding stabilizes the protein dimer (12).…”

Section: Resultsmentioning

confidence: 85%

“…The secondary structure prediction of McpS-LBR is not compatible with existing structural information on small-molecule binding domains, which prompted the resolution of its 3D structure. Among the chemoattractants identified for McpS, malate and succinate caused the strongest response (12). Therefore, McpS-LBR was crystallized in complex with these ligands, and the structures were solved using single-wavelength anomalous dispersion of selenomethionine derivatives.…”

Section: Resultsmentioning

confidence: 99%

“…The LBR of McpS (McpS-LBR) was predicted to contain two long and four short helices (12), which could not be shown to conform to the existing structural information on smallmolecule sensor domains. Recombinant McpS-LBR was found to recognize chemoattractants with affinities from 8 to 300 μM (12).…”

mentioning

confidence: 99%

“…To gain insight into the structure and action of cluster II receptors, we studied the McpS chemoreceptor of Pseudomonas putida KT2440, which was previously shown to mediate chemotaxis toward tricarboxylic acid (TCA) cycle intermediates and butyrate (12). The LBR of McpS (McpS-LBR) was predicted to contain two long and four short helices (12), which could not be shown to conform to the existing structural information on smallmolecule sensor domains.…”

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confidence: 99%

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Evidence for chemoreceptors with bimodular ligand-binding regions harboring two signal-binding sites

Pineda‐Molina

Reyes-Darías

Lacal

et al. 2012

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

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Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors are classified according to the size of their ligand-binding region. The well-studied cluster I proteins have a 100-to 150-residue ligand-binding region that contains a single site for chemoattractant recognition. Cluster II receptors, which contain a 220-to 300-residue ligand-binding region and which are almost as abundant as cluster I receptors, remain largely uncharacterized. Here, we report high-resolution structures of the ligand-binding region of the cluster II McpS chemotaxis receptor (McpS-LBR) of Pseudomonas putida KT2440 in complex with different chemoattractants. The structure of McpS-LBR represents a small-molecule binding domain composed of two modules, each able to bind different signal molecules. Malate and succinate were found to bind to the membrane-proximal module, whereas acetate binds to the membrane-distal module. A structural alignment of the two modules revealed that the ligand-binding sites could be superimposed and that amino acids involved in ligand recognition are conserved in both binding sites. Ligand binding to both modules was shown to trigger chemotactic responses. Further analysis showed that McpS-like receptors were found in different classes of proteobacteria, indicating that this mode of response to different carbon sources may be universally distributed. The physiological relevance of the McpS architecture may lie in its capacity to respond with high sensitivity to the preferred carbon sources malate and succinate and, at the same time, mediate lower sensitivity responses to the less preferred but very abundant carbon source acetate.sensor domain | four-helix bundle T he ability to sense and respond to extracellular signals is of crucial importance for microorganisms. Bacteria have several types of signal-transduction systems that sense environmental signals and trigger a corresponding response. Genome analyses indicate a dominant role for one-component systems, two-component systems, and chemoreceptor-based mechanisms in bacterial signal transduction (1, 2).Chemoreceptors have been initially described in the context of chemotactic signaling. However, more recent studies reveal that chemoreceptors are also involved in the regulation of different cellular processes, such as the synthesis of second messengers (3) or the control of gene expression during development (4). Typically, chemoreceptors are composed of a ligand-binding region (LBR) and a signaling domain. Signal recognition by the LBR creates a molecular stimulus that is conveyed to the signaling domain, which forms a complex with CheA and CheW. This molecular stimulus modulates CheA autophosphorylation and, subsequently, transphosphorylation toward the response regulator (5).The enterobacterial chemoreceptors, and in particular Tar and Tsr, have been studied extensively (5, 6). The Tar-LBR forms a four-helix bundle structure (7), and there are two mechanisms by which Tar-LBR recognizes chemoattractants. One mode consists o...

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Section: Resultssupporting

confidence: 66%

Section: Resultsmentioning

confidence: 85%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Evidence for chemoreceptors with bimodular ligand-binding regions harboring two signal-binding sites

Pineda‐Molina

Reyes-Darías

Lacal

et al. 2012

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

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Physiologically relevant divalent cations modulate citrate recognition by the McpS chemoreceptor

Lacal

García-Fontana

Callejo-García

et al. 2011

J of Molecular Recognition

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The McpS chemoreceptor of Pseudomonas putida KT2440 recognizes six different tricarboxylic acid (TCA) cycle intermediates. However, the magnitude of the chemotactic response towards these compounds differs largely, which has led to distinguish between strong attractants (malate, succinate, fumarate, oxaloacetate) and weak attractants (citrate, isocitrate). Citrate is abundantly present in plant tissues and root exudates and can serve as the only carbon source for growth. Citrate is known to form complexes with divalent cations which are also abundantly present in natural habitats of this bacterium. We have used isothermal titration calorimetry to study the formation of citrate-metal ion complexes. In all cases binding was entropy driven but significant differences in affinity were observed ranging from K(D)=157 µM (for Mg(2+)) to 3 µM (for Ni(2+)). Complex formation occurred over a range of pH and ionic strength. The ligand binding domain of McpS (McpS-LBD) was found to bind free citrate, but not complexes with physiologically relevant Mg(2+) and Ca(2+). In contrast, complexes with divalent cations which are present as trace elements (Co(2+), Cd(2+) and Ni(2+)) were recognized by McpS-LBD. This discrimination differs from other citrate sensing proteins. These results are discussed in the context of the three dimensional structure of free citrate and its complex with Mg(2+). Chemotaxis assays using P. putida revealed that taxis towards the strong attractant malate is strongly reduced in the presence of free citrate. However, this reduction is much less important in the presence of citrate-Mg(2+) complexes. The physiological relevance of these findings is discussed.

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Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

Ghai

Falconer

Collins

2011

J of Molecular Recognition

163

108

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Isothermal titration calorimetry (ITC) is a biophysical technique for measuring the formation and dissociation of molecular complexes and has become an invaluable tool in many branches of science from cell biology to food chemistry. By measuring the heat absorbed or released during bond formation, ITC provides accurate, rapid, and label-free measurement of the thermodynamics of molecular interactions. In this review, we survey the recent literature reporting the use of ITC and have highlighted a number of interesting studies that provide a flavour of the diverse systems to which ITC can be applied. These include measurements of protein-protein and protein-membrane interactions required for macromolecular assembly, analysis of enzyme kinetics, experimental validation of molecular dynamics simulations, and even in manufacturing applications such as food science. Some highlights include studies of the biological complex formed by Staphylococcus aureus enterotoxin C3 and the murine T-cell receptor, the mechanism of membrane association of the Parkinson's disease-associated protein α-synuclein, and the role of non-specific tannin-protein interactions in the quality of different beverages. Recent developments in automation are overcoming limitations on throughput imposed by previous manual procedures and promise to greatly extend usefulness of ITC in the future. We also attempt to impart some practical advice for getting the most out of ITC data for those researchers less familiar with the method.

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Identification of a Chemoreceptor for Tricarboxylic Acid Cycle Intermediates

Cited by 87 publications

References 57 publications

Evidence for chemoreceptors with bimodular ligand-binding regions harboring two signal-binding sites

Evidence for chemoreceptors with bimodular ligand-binding regions harboring two signal-binding sites

Physiologically relevant divalent cations modulate citrate recognition by the McpS chemoreceptor

Applications of isothermal titration calorimetry in pure and applied research—survey of the literature from 2010

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