Human EMG1 N1-specific pseudouridine methyltransferase, catalytic domain, in complex with S-adenosyl homocysteine

Target ID:

EMG1

Deposition Date:

Friday 11th December 2015

Families:

Transferases

Related Diseases:

Signalling

Structure type:

apo

Download Coordinates:

5FAI

Authors:

Dong A, Zeng H, Li Y, Bountra C, Arrowsmith CH, Edwards AM, Brown PJ, Wu H

Site:

Toronto

5FAI

tabs

Structure Details

The methylation of a variety of biologically active molecules is a common theme in biology and RNA methylation is one of them. RNA methylation has been observed in different species of RNA, including mRNAs, tRNAs, and non-coding RNAs. The functional roles of RNA methylation are not completely understood. Recent studies have indicated that RNA methylation could be an epigenetic mechanism for regulation of gene expression and other cellular process. The methylation of various RNA molecules is catalyzed by a variety of RNA mathyltransferases and the modification can occur at different positions in RNA molecules. N6-methyladenosine (m6A) and 5-methycytosine (5-mC) are the most common and abundant methylation modification in RNA molecules in eukaryotes. Studies have shown that m6A and 5-mC RNA methylation can affect RNA stability and mRNA translation.

Human EMG1 (essential for mitotic growth 1) is a highly conserved nucleolar protein involved in ribosome biogenesis. This protein methylates pseudouridine at position 1248 (Psi1248) in 18S rRNA and is involved in the biosynthesis of N1-methyl-N3-(3-amino-3-carboxypropyl) pseudouridine (m1acp3-Psi) that is conserved in eukaryotic 18S rRNA. In yeast, this protein plays an essential role in 40S ribosomal subunit biogenesis by facilitating the incorporation of ribosomal protein S19. A mutation in human EMG1 causes Bowen-Conradi syndrome, which is a lethal developmental disorder in newborns. However, the functional role of pseudouridine methylation is still not clear. We have solved the crystal structure of human EMG1 in complex with SAH at 1.8 Å resolution. EMG1 belongs to the SPOUT RNA methyltransferase family. The structure of EMG1 contains a signature a/b knot fold of SPOUT family, with the SAM-binding pocket sitting on top of the b-sheet. In addition, a citrate molecule (introduced by crystallization buffer) was also found near the SAM-binding site, which could mark the binding site of substrate RNA.

Materials and Methods

Target: EMG1

Entry clone source: MGC

Entry clone accession: GI:194328699

Construct (coding) sequence: gSGGEQAQDWDALPPKRPRLGAGNKIGGRRLIVVLEGASLETVKVGKTYELLNCDKHKSILLKNGRDPGEARPDITHQSLLMLMDSPLNRAGLLQVYIHTQKNVLIEVNPQTRIPRTFDRFCGLMVQLLHKLSVRAADGPQKLLKVIKNPVSDHFPVGCMKVGTSFSIPVVSDVRELVPSSDPIVFVVGAFAHGKVSVEYTEKMVSISNYPLSAALTCAKLTTAFEEVWGVI

Vector: pET28-MHL

Tags and additions: N-terminal: 6XHis-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQ*G

Host: E.coli BL21 (DE3) codon plus RIL (Stratagen).

Growth medium, induction protocol: EMG1 protein was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 μg/ml of kanamycin. Cells were grown at 37oC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15oC.

Extraction buffer, extraction method: Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80˚C. For purification, the cell paste was thawed and resuspended in lysis buffer buffer (50 mM HEPES, pH 7.4, 0.5 M NaCl, 5 mM imidazol, 2 mM β-mercaptoethanol, 5% glycerol) with protease inhibitor (1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.

Protein Purification: The crude extract was cleared by centrifugation. The lysate was loaded onto 5 ml HiTrap column (GE Healthcare), charged with Ni2+. The column was washed with 10 CV of 20 mM HEPES pH 7.4, containing 500 mM NaCl, 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was loaded on Superdex200 column (26x60) (GE Healthcare), equilibrated with 20 mM PIPES, pH 6.5, 250 mM NaCl. The fractions containing EMG1 were pooled and TEV protease was added to remove His-tag. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30S column (10x10) (GE Healthcare), equilibrated with buffer containing 20 mM PIPES, pH 6.5, and eluted with linear gradient of NaCl up to 500 mM concentration (20 CV).

Stock Concentration: 10.8 mg/mL

Enzymatic treatment: TEV

Mass spec characterization: The expected mass for EMG1 is 25333.5 Da, measured mass is 25333.3678 Da.

Crystallization: Purified EMG1 protein (4.9 mg/mL) was complexed with S-adenosyl-L-homocysteine (SAH) (Sigma) at 1:5 molar ratio of protein:SAH and crystallized using sitting drop vapor diffusion method at 20 °C by mixing 1 ml of the protein solution with 1 ml of the reservoir solution containing 20% PEG3350, 0.2M di-NH4 Citrate.