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Crystallization and preliminary X-ray crystallographic analysis of the oxysterol-binding protein Osh3 from Saccharomyces cerevisiae.

ABSTRACT: Oxysterol-binding protein (OSBP) related proteins (ORPs) are conserved from yeast to humans and are implicated in regulation of sterol homeostasis and in signal transduction pathways. Osh3 of Saccharomyces cerevisiae is a pleckstrin-homology (PH) domain-containing ORP member that regulates phosphoinositide metabolism at endoplasmic reticulum-plasma membrane contact sites. The N-terminal PH domain of Osh3 was purified and crystallized as a lysozyme fusion and the resulting crystal diffracted to 2.3?Å resolution. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a=98.03, b=91.31, c=84.13?Å, ?=81.41°. With two molecules in the asymmetric unit, the Matthews coefficient was 3.13?Å3?Da(-1). Initial attempts to solve the structure by molecular-replacement techniques using T4 lysozyme as a search model were successful. The C-terminal OSBP-related domain (OBD) of Osh3 was crystallized by the vapour-diffusion method and the resulting crystal diffracted to 1.5?Å resolution. The crystal was orthorhombic, belonging to space group P2(1)2(1)2(1), with unit-cell parameters a=41.57, b=87.52, c=100.58?Å. With one molecule in the asymmetric unit, the Matthews coefficient was 2.01?Å3?Da(-1). Initial attempts to solve the structure by the single-wavelength anomalous dispersion technique using bromine were successful.

SUBMITTER: Tong J

PROVIDER: S-EPMC3509973 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Crystallization and preliminary X-ray crystallographic analysis of the oxysterol-binding protein Osh3 from Saccharomyces cerevisiae.

Tong Junsen J Yang Huiseon H Ha Subin S Lee Youngjin Y Eom Soo Hyun SH Im Young Jun YJ

Acta crystallographica. Section F, Structural biology and crystallization communications 20121114 Pt 12

Oxysterol-binding protein (OSBP) related proteins (ORPs) are conserved from yeast to humans and are implicated in regulation of sterol homeostasis and in signal transduction pathways. Osh3 of Saccharomyces cerevisiae is a pleckstrin-homology (PH) domain-containing ORP member that regulates phosphoinositide metabolism at endoplasmic reticulum-plasma membrane contact sites. The N-terminal PH domain of Osh3 was purified and crystallized as a lysozyme fusion and the resulting crystal diffracted to 2 ...[more]

PMID: 23192032

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Project description:BackgroundMitochondria are unique organelles that are found in most eukaryotic cells. The main role of the mitochondria is to produce ATP. The nuclear genome encoded proteins Cbs1 and Cbs2 are located at the mitochondrial inner membrane and are reported to be essential for the translation of mitochondrial cytochrome b mRNA. Genetic studies show that Cbs2 protein recognizes the 5' untranslated leader sequence of mitochondrial cytochrome b mRNA. However, due to a lack of biochemical and structural information, this biological process remains unclear. To investigate the structural characteristics of how Saccharomyces cerevisiae (S. cerevisiae) Cbs2 tethers cytochrome b mRNA to the mitochondrial inner membrane, a preliminary X-ray crystallographic study was carried out and is reported here.MethodsThe target gene from S. cerevisiae was amplified by polymerase chain reaction. The PCR fragment was digested by the NdeI and XhoI restriction endonucleases and then inserted into expression vector p28. After sequencing, the plasmid was transformed into Escherichia coli C43 competent cells. The selenomethionine derivative Cbs2 protein was overexpressed using M9 medium based on a methionine-biosynthesis inhibition method. The protein was first purified to Ni2+-nitrilotriacetate affinity chromatography and then further purified by Ion exchange chromatography and Gel-filtration chromatography. The purified Se-Cbs2 protein was concentrated to 10 mg/mL. The crystallization trials were performed using the sitting-drop vapor diffusion method at 16 °C. The complete diffraction data was processed and scaled with the HKL2000 package and programs in the CCP4 package, respectively.ResultsCbs2 from S. cerevisiae was cloned, prokaryotic expressed and purified. The analysis of the size exclusion chromatography showed that the Cbs2 protein peaked at a molecular weight of approximately 90 KDa. The crystal belonged to the space group C2, with unit-cell parameters of a = 255.11, b = 58.10, c = 76.37, and β = 95.35°. X-ray diffraction data was collected at a resolution of 2.7 Å. The Matthews coefficient and the solvent content were estimated to be 3.22 Å 3 Da-1 and 61.82%, respectively.ConclusionsIn the present study Cbs2 from S. cerevisiae was cloned, expressed, purified, and crystallized for structural studies. The molecular weight determination results indicated that the biological assembly of Cbs2 may be a dimer.The preliminary X-ray crystallographic studies indicated the presence of two Cbs2 molecules in the asymmetric unit. This study will provide an experimental basis for exploring how Cbs2 protein mediates cytochrome b synthesis.

Dataset Information

Crystallization and preliminary X-ray crystallographic analysis of the oxysterol-binding protein Osh3 from Saccharomyces cerevisiae.

Publications

Crystallization and preliminary X-ray crystallographic analysis of the oxysterol-binding protein Osh3 from Saccharomyces cerevisiae.

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