The Molecular Replacement Solution and X-ray Refinement to 2.8 Å of a Decameric Complex of Human Cyclophilin A with the Immunosuppressive Drug Cyclosporin A

The enzyme catalase (H2O2–H2O2 oxidoreductase; E.C. 11.1.6) was purified from haemolysate of human placenta and crystallized using the vapour-diffusion technique. Synchrotron-radiation diffraction data have been collected to 1.76 Å resolution. The enzyme crystallized in the space group P212121, with unit-cell dimensions a = 83.6, b = 139.4, c = 227.5 Å. A molecular-replacement solution of the structure has been obtained using beef liver catalase (PDB code 4blc) as a search model.

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Crystallization and preliminary X-ray diffraction studies of frutalin, an α-D-galactose-specific lectin from Artocarpus incisa seeds

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15015186 ◽

2015 ◽

Vol 71 (10) ◽

pp. 1282-1285 ◽

Cited By ~ 3

Author(s):

Ana Cristina de Oliveira Monteiro-Moreira ◽

Humberto D'Muniz Pereira ◽

Antonio Eufrasio Vieira Neto ◽

Frederico Bruno Mendes Batista Moreno ◽

Marina Duarte Pinto Lobo ◽

...

Keyword(s):

Biomarker Discovery ◽

Spectrometric Analysis ◽

Carbohydrate Binding ◽

Molecular Replacement ◽

X Ray Diffraction ◽

X Ray ◽

Cell Parameters ◽

Maximum Resolution ◽

Replacement Solution ◽

Artocarpus Incisa

Frutalin is an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and is a powerful tool for tumour biomarker discovery. The crystallization and preliminary X-ray diffraction analysis of this lectin, which was isolated from Artocarpus incisa seeds, are reported here. Frutalin was purified and submitted to mass-spectrometric analysis. Diverse masses at approximately 16 kDa were observed in the deconvoluted spectra, which support the presence of isoforms. The best frutalin crystals were grown within a week in 0.1 M citric acid pH 3.5 which contained 25% PEG 3350 as a precipitant at 293 K, and diffracted to a maximum resolution of 1.81 Å. The monoclinic crystals belonged to space group I2, with unit-cell parameters a = 76.17, b = 74.56, c = 118.98 Å, β = 96.56°. A molecular-replacement solution was obtained which indicated the presence of four monomers per asymmetric unit. Crystallographic refinement of the structure is in progress.

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Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophileHalothermothrix orenii

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309111041091 ◽

2012 ◽

Vol 68 (2) ◽

pp. 240-243 ◽

Cited By ~ 2

Author(s):

Lokesh D. Kori ◽

Andreas Hofmann ◽

Bharat K. C. Patel

Keyword(s):

Metal Ion ◽

Molecular Replacement ◽

Asymmetric Unit ◽

X Ray Diffraction ◽

Unit Cell Parameters ◽

Orthorhombic Space Group ◽

X Ray ◽

Cell Parameters ◽

Replacement Solution ◽

Metal Ion Affinity Chromatography

A ribokinase gene (rbk) from the anaerobic halothermophilic bacteriumHalothermothrix oreniiwas cloned and overexpressed inEscherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 45.6,b= 61.1,c= 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.

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Crystallization and preliminary X-ray analysis of a highly stable novel SGNH hydrolase (Est24) fromSinorhizobium meliloti

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x13033918 ◽

2014 ◽

Vol 70 (2) ◽

pp. 193-195 ◽

Cited By ~ 2

Author(s):

Bum Han Ryu ◽

Duy Duc Nguyen ◽

Tri Duc Ngo ◽

Changsuk Oh ◽

Ramesh Pandian ◽

...

Keyword(s):

Sinorhizobium Meliloti ◽

Structure Refinement ◽

Ammonium Phosphate ◽

Molecular Replacement ◽

X Ray Diffraction ◽

X Ray ◽

Cell Parameters ◽

Replacement Solution ◽

Hydrolysis Of ◽

Hydrolase Family

The SGNH hydrolase family includes enzymes that catalyze the hydrolysis of a broad range of substrates. Here, the crystallization and preliminary X-ray crystallographic studies of a novel SGNH hydrolase (Est24) fromSinorhizobium melilotiwere performed. Recombinant Est24 protein containing an N-terminal His tag was expressed inEscherichia coliand purified to homogeneity. Est24 was then crystallized using a solution consisting of 0.2 Mammonium phosphate pH 4.6, 20% polyethylene glycol 3350. X-ray diffraction data were collected to a resolution of 1.45 Å with anRmergeof 9.4%. The Est24 crystals belonged to space groupC2, with unit-cell parametersa= 129.09,b = 88.63,c= 86.15 Å, α = 90.00, β = 114.30, γ = 90.00°. A molecular-replacement solution was obtained using the crystal structure ofMycobacterium smegmatisarylesterase as a template and structure refinement of Est24 is in progress.

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Expression, purification, crystallization and preliminary X-ray diffraction analysis ofAspergillus terreusendo-β-1,4-glucanase from glycoside hydrolase family 12

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x13034936 ◽

2014 ◽

Vol 70 (2) ◽

pp. 267-270 ◽

Cited By ~ 5

Author(s):

Fernando Segato ◽

Gabriela L. Berto ◽

Evandro Ares de Araújo ◽

João Renato Muniz ◽

Igor Polikarpov

Keyword(s):

Glycoside Hydrolase ◽

Aspergillus Terreus ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Molecular Replacement ◽

X Ray Diffraction ◽

X Ray ◽

Cell Parameters ◽

Replacement Solution ◽

Hydrolase Family

Endoglucanases are important enzymes that are involved in the modification and degradation of cellulose. Filamentous fungi such asAspergillus terreusare effective biomass degraders in nature owing to their capacity to produce an enzymatic arsenal of glycoside hydrolases, including endoglucanase from glycoside hydrolase family 12 (GH12). TheA. terreusGH12 endoglucanase was cloned and overexpressed inA. nidulans, purified and crystallized. A single crystal was obtained from a solution consisting of 2 Mammonium sulfate, 5%(v/v) 2-propanol. X-ray diffraction data were collected to a resolution of 1.85 Å using synchrotron radiation and a preliminary molecular-replacement solution was obtained in the trigonal space groupP3221. The unit-cell parameters werea=b= 103.24,c= 48.96 Å.

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Crystallization, preliminary X-ray analysis and molecular-replacement solution of the carboxy form of haemoglobin I from the fishBrycon cephalus

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444900015262 ◽

2000 ◽

Vol 56 (12) ◽

pp. 1685-1687 ◽

Cited By ~ 1

Author(s):

R. T. Honda ◽

P. Delatorre ◽

V. Fadel ◽

F. Canduri ◽

M. Dellamano ◽

...

Keyword(s):

Molecular Replacement ◽

X Ray ◽

Replacement Solution

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Crystallization and preliminary X-ray diffraction studies of piratoxin III, a D-49 phospholipase A2 from the venom of Bothrops pirajai

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444999004576 ◽

1999 ◽

Vol 55 (6) ◽

pp. 1229-1230 ◽

Cited By ~ 12

Author(s):

Lee Wen-Hwa ◽

Marcos H. Toyama ◽

Andreimar M. Soares ◽

José R. Giglio ◽

Sérgio Marangoni ◽

...

Keyword(s):

Search Model ◽

Molecular Replacement ◽

X Ray Diffraction ◽

Unit Cell Parameters ◽

X Ray ◽

Cell Parameters ◽

Diffusion Technique ◽

Vapour Diffusion ◽

Replacement Solution ◽

Bothrops Pirajai

Piratoxin III (PrTX-III) is a phospholipase A2 (PLA2, E.C. 3.1.1.4, phosphatide sn-2 acylhydrolase) isolated from Bothrops pirajai. Crystals of PrTX-III were obtained using the vapour-diffusion technique and X-ray diffraction data have been collected to 2.7 Å resolution. The enzyme was crystallized in the space group C2 with unit-cell parameters a = 60.88, b = 100.75, c = 48.19 Å, β = 123.89°. A molecular-replacement solution of the structure has been found using bothropstoxin I from the venom of B. jararacussu as a search model.

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Cyclophilin A regulates A549 cells apoptosis via stabilizing Twist1 protein

Journal of Cell Science ◽

10.1242/jcs.259018 ◽

2021 ◽

Author(s):

Yaru Wu ◽

Zhenling Ma ◽

Yanyan Zhang ◽

Min Zhang ◽

Wenwen Zhang ◽

...

Keyword(s):

Cyclosporin A ◽

Intracellular Signaling ◽

A549 Cells ◽

Cyclophilin A ◽

Immunosuppressive Drug ◽

Nuclear Accumulation ◽

Biological Processes ◽

Peptide Bonds ◽

Intracellular Target

Cyclophilin A (CypA) is an essential member of the immunophilin family. As an intracellular target of immunosuppressive drug cyclosporin A (CsA) or a peptidyl-prolyl cis/trans isomerase (PPIase), it catalyzes the cis-trans isomerization of proline amidic peptide bonds, through which, it regulates a variety of biological processes, such as intracellular signaling, transcription, and apoptosis. In this study, we found that intracellular CypA enhanced Twist1 phosphorylation at Ser68 and inhibited apoptosis in A549 cells. Mechanistically, CypA could mediate the phosphorylation of Twist1 at Ser68 via p38 MAPK, which inhibited its ubiquitination-mediated degradation. In addition, CypA increased Twist–p65 interaction and nuclear accumulation, which regulated Twist1-dependent expression of CDH1 and CDH2. Our findings collectively indicated the role of CypA in Twist1-mediated A549 cells apoptosis through stabilizing Twist1 protein.

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