The periplasmic sensing domain of &lt;i&gt;Pseudomonas fluorescens &lt;/i&gt;chemotactic transducer of amino acids type B (CtaB): Cloning, refolding, purification, crystallization, and X-ray crystallographic analysis

Ud-Din, Abu Iftiaf Md Salah; Roujeinikova, Anna

doi:10.5582/bst.2016.01218

Cited by 2 publications

(1 citation statement)

References 27 publications

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“…pylori TlpC (this study), Tlp1 and Tlp3 from C . jejuni 23 , 24 , CtaA and CtaB from Pseudomonas fluorescens 89 , 90 , VfcA from Vibrio fischeri 91 , and PctA from P . putida 92 , are monomeric in solution.…”

Section: Discussionmentioning

confidence: 99%

Helicobacter pylori chemoreceptor TlpC mediates chemotaxis to lactate

Machuca

Johnson

Liu

et al. 2017

Sci Rep

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It is recently appreciated that many bacterial chemoreceptors have ligand-binding domains (LBD) of the dCACHE family, a structure with two PAS-like subdomains, one membrane-proximal and the other membrane-distal. Previous studies had implicated only the membrane-distal subdomain in ligand recognition. Here, we report the 2.2 Å resolution crystal structure of dCACHE LBD of the Helicobacter pylori chemoreceptor TlpC. H. pylori tlpC mutants are outcompeted by wild type during stomach colonisation, but no ligands had been mapped to this receptor. The TlpC dCACHE LBD has two PAS-like subdomains, as predicted. The membrane-distal one possesses a long groove instead of a small, well-defined pocket. The membrane-proximal subdomain, in contrast, had a well-delineated pocket with a small molecule that we identified as lactate. We confirmed that amino acid residues making contact with the ligand in the crystal structure—N213, I218 and Y285 and Y249—were required for lactate binding. We determined that lactate is an H. pylori chemoattractant that is sensed via TlpC with a K D = 155 µM. Lactate is utilised by H. pylori, and our work suggests that this pathogen seeks out lactate using chemotaxis. Furthermore, our work suggests that dCACHE domain proteins can utilise both subdomains for ligand recognition.

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Section: Discussionmentioning

confidence: 99%

Helicobacter pylori chemoreceptor TlpC mediates chemotaxis to lactate

Machuca

Johnson

Liu

et al. 2017

Sci Rep

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Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Machuca

Roujeinikova

2017

JoVE

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Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

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The periplasmic sensing domain of <i>Pseudomonas fluorescens </i>chemotactic transducer of amino acids type B (CtaB): Cloning, refolding, purification, crystallization, and X-ray crystallographic analysis

Cited by 2 publications

References 27 publications

Helicobacter pylori chemoreceptor TlpC mediates chemotaxis to lactate

Helicobacter pylori chemoreceptor TlpC mediates chemotaxis to lactate

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

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