method-for-efficient-refolding-purification-chemoreceptor-ligand

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Monash Biomedicine Discovery Institute

Monash University

Cytotoxin associated gene product A (CagA) is an oncogenic protein secreted by the gastric bacterium Helicobacter pylori. Internalization of CagA by human epithelial cells occurs by an unknown mechanism that requires interaction with the host membrane lipid phosphatidylserine.

Phospholipid binding residues of eukaryotic membrane-remodelling F-BAR domain proteins are conserved in Helicobacter pylori CagA.

Helicobacter pylori infections are associated with gastritis, duodenal and gastric ulcers and gastric adenocarcinoma. Bacterial chemotaxis, mediated by four different chemoreceptors (also termed transducer-like proteins (Tlp)), plays an important role in initial colonization and development of disease. Chemoreceptor sensory domains of H. pylori share no significant sequence similarity with those of Escherichia coli or any other non-Epsilonproteobacteria. The structural basis of how chemical signals are recognized by chemoreceptors of H. pylori is poorly understood mainly due to the lack of a robust procedure to purify their sensory domains in a soluble form. This study reports a method for extraction of the periplasmic sensory domain of transducer-like protein C (TlpC) from inclusion bodies and refolding to yield 5mg pure crystallizable protein per 1l of bacterial culture. Purified protein was monomeric in solution by size-exclusion chromatography and folded according to the circular dichroism spectrum. Crystals have been grown by the hanging-drop vapor-diffusion method using PEG 4000 as a precipitating agent. The crystals belonged to space group C2, with unit-cell parameters a=189.3, b=103.2, c=61.8Å, β=98.3. A complete X-ray diffraction data set has been collected to 2.2 Å resolution using cryocooling conditions and synchrotron radiation. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains three monomers.

Expression, refolding, purification and crystallization of the sensory domain of the TlpC chemoreceptor from Helicobacter pylori for structural studies.

The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases.

Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase.

Chemotaxis and motility play an important role in the colonisation of avian and human hosts by Campylobacter jejuni. Chemotactic recognition of extracellular signals is mediated by the periplasmic sensing domain of methyl-accepting chemotactic proteins (membrane-embedded receptors). In this work, we report a high-resolution structure of the periplasmic sensing domain of transducer-like protein 1 (Tlp1), an aspartate receptor of C. jejuni. Crystallographic analysis revealed that it contains two Per-Arnt-Sim (PAS) subdomains. An acetate and chloride ions (both from the crystallisation buffer) were observed bound to the membrane-proximal and membrane-distal PAS subdomains, respectively. Surprisingly, despite being crystallised in the presence of aspartate, the structure did not show any electron density corresponding to this amino acid. Furthermore, no binding between the sensing domain of Tlp1 and aspartate was detected by microcalorimetric experiments. These structural and biophysical data suggest that Tlp1 does not sense aspartate directly; instead, ligand recognition is likely to occur indirectly via an as yet unidentified periplasmic binding protein.

The crystal structure of the tandem-PAS sensing domain of Campylobacter jejuni chemoreceptor Tlp1 suggests indirect mechanism of ligand recognition.

Chemotaxis towards nutrients plays a crucial role in root colonization by Pseudomonas fluorescens. The P. fluorescens chemotactic transducer of amino acids type A (CtaA) mediates movement towards amino acids present in root exudates. In this study, the periplasmic sensory domain of CtaA has been crystallized by the hanging-drop vapor diffusion method using ammonium sulfate as a precipitating agent. A complete data set was collected to 1.9 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belong to space group I222 or I212121, with unit-cell parameters a = 67.2, b = 76.0, c = 113.3 Å. This is an important step towards elucidation of the structural basis of how CtaA recognizes its signal molecules and transduces the signal across the membrane.

Cloning, purification, crystallization and X-ray crystallographic analysis of the periplasmic sensing domain of Pseudomonas fluorescens chemotactic transducer of amino acids type A (CtaA).

General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections.

Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT).

α-Carbonic anhydrase of Helicobacter pylori (HpαCA) plays an important role in the acclimation of this oncobacterium to the acidic pH of the stomach. Sulfonamide inhibitors of HpαCA possess anti-H. pylori activity. The crystal structures of complexes of HpαCA with a family of acetazolamide-related sulfonamides have been determined. Analysis of the structures revealed that the mode of sulfonamide binding correlates well with their inhibitory activities. In addition, comparisons with the corresponding inhibitor complexes of human carbonic anhydrase II (HCAII) indicated that HpαCA possesses an additional, alternative binding site for sulfonamides that is not present in HCAII. Furthermore, the hydrophobic pocket in HCAII that stabilizes the apolar moiety of sulfonamide inhibitors is replaced with a more open, hydrophilic pocket in HpαCA. Thus, our analysis identified major structural features can be exploited in the design of selective and more potent inhibitors of HpαCA that may lead to novel antimicrobials.

Structure-Activity Relationship for Sulfonamide Inhibition of Helicobacter pylori α-Carbonic Anhydrase.

Pseudomonas fluorescens is a plant growth promoting rhizobacterium that provides nutrients for growth and induces systemic resistance against plant diseases. It has been linked with a number of human diseases including nosocomial infections and bacterial cystitis. Chemotactic motility of P. fluorescens towards root exudates plays a crucial role in establishing a symbiotic relationship with host plants. The P. fluorescens chemotactic transducer of amino acids type B (CtaB) mediates chemotaxis towards amino acids. As a step towards elucidation of the structural basis of ligand recognition by CtaB, we have produced crystals of its recombinant sensory domain and performed their X-ray diffraction analysis. The periplasmic sensory domain of CtaB has been expressed, purified, and crystallized by the hanging-drop vapor diffusion method using ammonium sulfate as a precipitating agent. A complete data set was collected to 2.2 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belong to space group P212121, with unit-cell parameters a = 34.5, b = 108.9, c = 134.6 Å. Calculation of the Matthews coefficient and the self-rotation function using this data set suggested that the asymmetric unit contains a protein dimer. Detailed structural analysis of CtaB would be an important step towards understanding the molecular mechanism underpinning the recognition of environmental signals and transmission of the signals to the inside of the cell.

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

Chemotaxis is the directed motility by means of which microbes sense chemical cues and relocate towards more favorable environments. Methyl-accepting chemotaxis proteins (MCPs) are the most common receptors in bacteria and archaea. They are arranged as trimers of dimers that, in turn, form hexagonal arrays in the cytoplasmic membrane or in the cytoplasm. Several different classes of MCPs have been identified according to their ligand binding region and membrane topology. MCPs have been further classified based on the length and sequence conservation of their cytoplasmic domains. Clusters of membrane-embedded MCPs often localize to the poles of the cell, whereas cytoplasmic MCPs can be targeted to the poles or distributed throughout the cell body. MCPs play an important role in cell survival, pathogenesis, and biodegradation. Bacterial adaptation to diverse environmental conditions promotes diversity among the MCPs. This review summarizes structure, classification, and structure-activity relationship of the known MCP receptors, with a brief overview of the signal transduction mechanisms in bacteria and archaea.

Methyl-accepting chemotaxis proteins: a core sensing element in prokaryotes and archaea.

Cytotoxin-associated gene product A (CagA) is a major virulence factor secreted by Helicobacter pylori. CagA activity in the gastric epithelium is associated with higher risk of gastric cancer development. Bacterial type IV secretion system (T4SS)-mediated translocation of CagA into the cytosol of human epithelial cells occurs via a poorly understood mechanism that requires CagA interaction with the host membrane lipid phosphatidylserine (PS) and host cell receptor integrin α₅β₁. Here we have characterized the isolated recombinant middle fragment of CagA (CagA-M) that contains the positively-charged PS-binding region (aa 613-636) and a putative β₁ integrin binding site, but lacks the EPIYA region, secretion signal peptide and the CagA multimerization motif. We show that CagA-M, when immobilized on latex beads, is capable of binding to, and triggering its own uptake into, gastric epithelial cells in the absence of infection with cagA-positive H. pylori. Using site-directed mutagenesis, fluorescent and electron microscopy, and highly-specific inhibitors, we demonstrate that the cell-binding and endocytosis-like internalization of CagA-M are dependent on (1) binding to PS; (2) β₁ integrin activity; and (3) actin dynamics. Interaction of CagA-M with the host cells is accompanied by the development of long filopodia-like protrusions (macrospikes). This novel morphology is different from the hummingbird phenotype induced by the translocation of full-length CagA. The determinants within CagA-M and within the host that are important for endocytosis-like internalization into host cells are very similar to those observed for T4SS-mediated internalization of full-length CagA, suggesting that the latter may involve an endocytic pathway.

The Middle Fragment of Helicobacter pylori CagA Induces Actin Rearrangement and Triggers Its Own Uptake into Gastric Epithelial Cells.

Rotation of the bacterial flagellum is powered by a proton influx through the peptidoglycan (PG)-tethered stator ring MotA/B. MotA and MotB form an inner-membrane complex that does not conduct protons and does not bind to PG until it is inserted into the flagellar motor. The opening of the proton channel involves association of the plug helices in the periplasmic region of the MotB dimer into a parallel coiled coil. Here, we have characterised the structure of a soluble variant of full-length Helicobacter pylori MotB in which the plug helix was engineered to be locked in a parallel coiled coil state, mimicking the open state of the stator. Fluorescence resonance energy transfer measurements, combined with PG-binding assays and fitting of the crystal structures of MotB fragments to the small angle X-ray scattering (SAXS) data revealed that the protein's C-terminal domain has a PG-binding-competent conformation. Molecular modelling against the SAXS data suggested that the linker in H. pylori MotB forms a subdomain between the plug and the C-terminal domain, that 'clamps' the coiled coil of the plug, thus stabilising the activated form of the protein. Based on these results, we present a pseudo-atomic model structure of full-length MotB in its activated form.

Structural analysis of variant of Helicobacter pylori MotB in its activated form, engineered as chimera of MotB and leucine zipper.

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.

Helicobacter pylori chemoreceptor TlpC mediates chemotaxis to lactate.

Many pathogenic bacteria require flagella-mediated motility to colonise and persist in their hosts. Helicobacter pylori and Campylobacter jejuni are flagellated epsilonproteobacteria associated with several human pathologies, including gastritis, acute diarrhea, gastric carcinoma and neurological disorders. In both species, glycosylation of flagellin with an unusual sugar pseudaminic acid (Pse) plays a crucial role in the biosynthesis of functional flagella, and thereby in bacterial motility and pathogenesis. Pse is found only in pathogenic bacteria. Its biosynthesis via six consecutive enzymatic steps has been extensively studied in H. pylori and C. jejuni. This review highlights the importance of flagella glycosylation and details structural insights into the enzymes in the Pse pathway obtained via a combination of biochemical, crystallographic, and mutagenesis studies of the enzyme-substrate and -inhibitor complexes. It is anticipated that understanding the underlying structural and molecular basis of the catalytic mechanisms of the Pse-synthesising enzymes will pave the way for the development of novel antimicrobials.

Anna Roujeinikova

Infection and Immunity Program

Monash Biomedicine Discovery Institute

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Anna Roujeinikova

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Infection and Immunity Program

Monash Biomedicine Discovery Institute

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Infection and Immunity Program