Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Preliminary crystallographic analysis of two oligomerization-deficient mutants of the aerolysin toxin, H132D and H132N, in their proteolyzed forms.

ABSTRACT: Aerolysin is a major virulence factor produced by the Gram-negative bacterium Aeromonas hydrophila and is a member of the ?-pore-forming toxin family. Two oligomerization-deficient aerolysin mutants, H132D and H132N, have been overproduced, proteolyzed by trypsin digestion and purified. Crystals were grown from the proteolyzed forms and diffraction data were collected for the two mutants to 2.1 and 2.3?Å resolution, respectively. The prism-shaped crystals belonged to space group C2. The crystal structure of the mutants in the mature, but not heptameric, aerolysin form will provide insight into the intermediate states in the oligomerization process of a pore-forming toxin.

SUBMITTER: Pernot L

PROVIDER: S-EPMC2998370 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Preliminary crystallographic analysis of two oligomerization-deficient mutants of the aerolysin toxin, H132D and H132N, in their proteolyzed forms.

Pernot Lucile L Schiltz Marc M van der Goot F Gisou FG

Acta crystallographica. Section F, Structural biology and crystallization communications 20101126 Pt 12

Aerolysin is a major virulence factor produced by the Gram-negative bacterium Aeromonas hydrophila and is a member of the β-pore-forming toxin family. Two oligomerization-deficient aerolysin mutants, H132D and H132N, have been overproduced, proteolyzed by trypsin digestion and purified. Crystals were grown from the proteolyzed forms and diffraction data were collected for the two mutants to 2.1 and 2.3 Å resolution, respectively. The prism-shaped crystals belonged to space group C2. The crystal ...[more]

PMID: 21139211

Similar Datasets

Preliminary X-ray crystallographic studies on acetate kinase (AckA) from Salmonella typhimurium in two crystal forms.

Project description:Acetate kinase (AckA) catalyzes the reversible transfer of a phosphate group from acetyl phosphate to ADP, generating acetate and ATP, and plays a central role in carbon metabolism. In the present work, the gene corresponding to AckA from Salmonella typhimurium (StAckA) was cloned in the IPTG-inducible pRSET C vector, resulting in the attachment of a hexahistidine tag to the N-terminus of the expressed enzyme. The recombinant protein was overexpressed, purified and crystallized in two different crystal forms using the microbatch-under-oil method. Form I crystals diffracted to 2.70 Å resolution when examined using X-rays from a rotating-anode X-ray generator and belonged to the monoclinic space group C2, with unit-cell parameters a = 283.16, b = 62.17, c = 91.69 Å, ? = 93.57°. Form II crystals, which diffracted to a higher resolution of 2.35 Å on the rotating-anode X-ray generator and to 1.90 Å on beamline BM14 of the ESRF, Grenoble, also belonged to space group C2 but with smaller unit-cell parameters (a = 151.01, b = 78.50, c = 97.48 Å, ? = 116.37°). Calculation of Matthews coefficients for the two crystal forms suggested the presence of four and two protomers of StAckA in the asymmetric units of forms I and II, respectively. Initial phases for the form I diffraction data were obtained by molecular replacement using the coordinates of Thermotoga maritima AckA (TmAckA) as the search model. The form II structure was phased using a monomer of form I as the phasing model. Inspection of the initial electron-density maps suggests dramatic conformational differences between residues 230 and 300 of the two crystal forms and warrants further investigation.

| S-EPMC3232164 | biostudies-literature

Dual chaperone role of the C-terminal propeptide in folding and oligomerization of the pore-forming toxin aerolysin.

Project description:Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs). PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP). By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function.

| S-EPMC3136475 | biostudies-literature

Crystallization and preliminary crystallographic analysis of Achromobacter protease I mutants.

Project description:Achromobacter protease I (API), a serine protease, shows an order of magnitude higher activity than bovine trypsin. The optimum pH of mutant enzymes with His210 replaced by Ser (H210S) and Trp169 replaced by Phe (W169F) has been shown to shift from approximately pH 9 (wild-type enzyme) to approximately pH 7 while retaining high activity. The mutants were crystallized by the hanging-drop vapour-diffusion technique with 2 M ammonium sulfate as the precipitant. The space group of the W169F mutant crystal was P1, with unit-cell parameters a = 42.6, b = 34.7, c = 69.5 Å, α = 91.8, β = 97.5, γ = 89.9°, while the space group of the H210S mutant crystal was P2(1), with unit-cell parameters a = 42.4, b = 34.2, c = 67.7 Å, β = 97.6°. Diffraction data were collected from W169F and H210S crystals to resolutions of 2.0 and 2.3 Å, respectively.

| S-EPMC3001667 | biostudies-literature

Purification, crystallization and preliminary crystallographic studies of haemoglobin from mongoose (Helogale parvula) in two different crystal forms induced by pH variation.

Project description:Haemoglobin (Hb) is a respiratory pigment; it is a tetrameric protein that ferries oxygen from the lungs to tissues and transports carbon dioxide on the return journey. The oxygen affinity of haemoglobin is regulated by the concentration of oxygen surrounding it and several efforts have revealed the shapes of Hb in different states and with different functions. However, study of the molecular basis of Hbs from low-oxygen-affinity species is critically needed in order to increase the understanding of the mechanism behind oxygen adaptation. The present study reports the preliminary crystallographic study of low-oxygen-affinity haemoglobin from mongoose, a burrowing mammal. Haemoglobin from mongoose was purified by anion-exchange chromatography, crystallized using the hanging-drop vapour-diffusion method and diffraction data sets were collected from monoclinic (2.3 Å resolution) and orthorhombic (2.9 Å resolution) crystal forms obtained by pH variation. The monoclinic and orthorhombic asymmetric units contained half and a whole biological molecule, respectively.

| S-EPMC3564612 | biostudies-literature

Purification, crystallization and preliminary crystallographic study of low oxygen-affinity haemoglobin from cat (Felis silvestris catus) in two different crystal forms.

Project description:Haemoglobin is a metalloprotein which plays a major role in the transportation of oxygen from the lungs to tissues and of carbon dioxide back to the lungs. The present work reports the preliminary crystallographic study of low oxygen-affinity haemoglobin from cat in different crystal forms. Cat blood was collected, purified by anion-exchange chromatography and crystallized in two different conditions by the hanging-drop vapour-diffusion method under unbuffered low-salt and buffered high-salt concentrations using PEG 3350 as a precipitant. Intensity data were collected using MAR345 and MAR345dtb image-plate detector systems. Cat haemoglobin crystallizes in monoclinic and orthorhombic crystal forms with one and two whole biological molecules (alpha(2)beta(2)), respectively, in the asymmetric unit.

| S-EPMC2650469 | biostudies-literature

Preliminary crystallographic analysis of recombinant VapBC-15 toxin-antitoxin complex from Mycobacterium tuberculosis.

Project description:The Mycobacterium tuberculosis vapBC15 locus encodes a toxin-antitoxin complex. VapC-15 is a toxin and possesses ribonuclease activity and VapB-15 is an antitoxin which both binds and inhibits the VapC-15 toxin. In this study, vapBC15 genes were cloned and co-expressed in Escherichia coli. The complex was purified to homogeneity by affinity and size-exclusion chromatography. The VapBC-15 complex was crystallized using the sitting-drop vapour-diffusion technique. The crystals diffracted to 2.6 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 85.63, b = 139.09, c = 148.86 Å. The self-rotation function combined with Matthews coefficient and solvent-content calculations suggests the presence of either six or eight molecules of the complex in the asymmetric unit.

| S-EPMC3818043 | biostudies-literature

Crystallization and preliminary crystallographic analysis of two eukaryotic fructosyl peptide oxidases.

Project description:Fructosyl peptide oxidase (FPOX) catalyses the oxidation of ?-glycated dipeptides such as N(?)-(1-deoxy-D-fructos-1-yl)-L-valyl-L-histidine (Fru-ValHis) and is used in the diagnosis of diabetes mellitus. Here, two thermostable mutants of FPOX, CFP-T7 and EFP-T5M, were crystallized by the sitting-drop vapour-diffusion method. The crystal of CFP-T7 belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 110.09, c = 220.48 Å, and that of EFP-T5M belonged to the monoclinic space group P2(1), with unit-cell parameters a = 43.00, b = 230.05, c = 47.27 Å, ? = 116.99°. The crystals of CFP-T7 and EFP-T5M diffracted to 1.8 and 1.6 Å resolution, respectively.

| S-EPMC3564613 | biostudies-literature

Preliminary crystallographic analysis of two hypothetical ribose-5-phosphate isomerases from Streptococcus mutans.

Project description:Study of the enzymes from sugar metabolic pathways may provide a better understanding of the pathogenesis of the human oral pathogen Streptococcus mutans. Bioinformatics, biochemical and crystallization methods were used to characterize and understand the function of two putative ribose-5-phosphate isomerases: SMU1234 and SMU2142. The proteins were cloned and constructed with N-terminal His tags. Protein purification was performed by Ni(2+)-chelating and size-exclusion chromatography. The crystals of SUM1234 diffracted to 1.9?Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 48.97, b = 98.27, c = 101.09?Å, ? = ? = ? = 90°. The optimized SMU2142 crystals diffracted to 2.7?Å resolution and belonged to space group P1, with unit-cell parameters a = 53.7, b = 54.1, c = 86.5?Å, ? = 74.2, ? = 73.5, ? = 83.7°. Initial phasing of both proteins was attempted by molecular replacement; the structure of SMU1234 could easily be solved, but no useful results were obtained for SMU2142. Therefore, SeMet-labelled SMU2142 will be prepared for phasing.

| S-EPMC3374514 | biostudies-literature

A preliminary crystallographic investigation of avidin.

Project description:Avidin was crystallized in various forms from 3m-ammonium sulphate or sodium phosphate at pH5.2. X-ray-crystallographic analysis of crystals from phosphate showed them to be orthorhombic, space group C222, containing two molecules per unit cell and with a single subunit as the asymmetric unit. The formation of the complex with biotin did not affect the morphology of the crystals.

| S-EPMC1179081 | biostudies-other

Structural, physicochemical and dynamic features conserved within the aerolysin pore-forming toxin family.

Project description:Aerolysin is the founding member of a major class of β-pore-forming toxins (β-PFTs) found throughout all kingdoms of life. PFTs are cytotoxic proteins produced as soluble monomers, which oligomerize at the membrane of target host cells forming pores that may lead to osmotic lysis and cell death. Besides their role in microbial infection, they have become interesting for their potential as biotechnological sensors and delivery systems. Using an approach that integrates bioinformatics with molecular modeling and simulation, we looked for conserved features across this large toxin family. The cell surface-binding domains present high variability within the family to provide membrane receptor specificity. On the contrary, the novel concentric double β-barrel structure found in aerolysin is highly conserved in terms of sequence, structure and conformational dynamics, which likely contribute to preserve a common transition mechanism from the prepore to the mature pore within the family.Our results point to the key role of several amino acids in the conformational changes needed for oligomerization and further pore formation, such as Y221, W227, P248, Q263 and L277, which we propose are involved in the release of the stem loop and the two adjacent β-strands to form the transmembrane β-barrel.

| S-EPMC5654971 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data