Human methyltransferase like 3 - 14 dimer in complex with cofactor

Target ID:

METTL3-METTL14

Deposition Date:

Friday 30th September 2016

Families:

Methyltransferases

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

5TEY

Authors:

Dong A, Zeng H, Li Y, Tempel W, Bountra, C., Arrowsmith, C.H., Edwards, A.M., Brown PJ, Wu H

Site:

Toronto

5TEY

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 methyltransferases 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.

In human, m6A methylation is catalyzed by two proteins: METTL3 and METTL14. These two enzymes belong to class I methyltransferase family. Individually, both proteins exhibit weak methyltransferase activity. But the METTL3-METTLE14 complex has much higher catalytic activity. We have solved the crystal structure of METTL3-METTL14 heterodimer at 1.8 Å resolution. Both METTL3 and METTL14 adopt a class I methyltransferase fold. However, only one molecule of SAH was found in METTL3 active site. A large interface (2,460 Å2) between METTL3 and METTLE14 was formed by extensive hydrogen bonds. A positively charged groove at the interface area could be the potential substrate RNA binding site.

Materials and Methods

METTL3-METTL14

PDB:5TEY

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:METTL3:BC007449; METTL14:BC001650
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQ*G
Host:Sf9

Construct

Prelude:
Sequence:METTL3: MSDTWSSIQAHKKQLDSLRERLQRRRKQDSGHLDLRNPEAALSPTFRSDSPVPTAPTSGGPKPSTASAVPELATDPELEKKLLHHLSDLALTLPTDAVSICLAISTPDAPATQDGVESLLQKFAAQELIEVKRGLLQDDAHPTLVTYADHSKLSAMMGAVAEKKGPGEVAGTVTGQKRRAEQDSTTVAAFASSLVSGLNSSASEPAKEPAKKSRKHAASDVDLEIESLLNQQSTKEQQSKKVSQEILELLNTTTAKEQSIVEKFRSRGRAQVQEFCDYGTKEECMKASDADRPCRKLHFRRIINKHTDESLGDCSFLNTCFHMDTCKYVHYEIDACMDSEAPGSKDHTPSQELALTQSVGGDSSADRLFPPQWICCDIRYLDVSILGKFAVVMADPPWDIHMELPYGTLTDDEMRRLNIPVLQDDGFLFLWVTGRAMELGRECLNLWGYERVDEIIWVKTNQLQRIIRTGRTGHWLNHGKEHCLVGVKGNPQGFNQGLDCDVIVAEVRSTSHKPDEIYGMIERLSPGTRKIELFGRPHNVQPNWITLGNQLDGIHLLDPDVVARFKQKYPDGIISKPKNL - METTL14:MDSRLQEIRERQKLRRQLLAQQLGAESADSIGAVLNSKDEQREIAETRETCRASYDTSAPNAKRKYLDEGETDEDKMEEYKDELEMQQDEENLPYEEEIYKDSSTFLKGTQSLNPHNDYCQHFVDTGHRPQNFIRDVGLADRFEEYPKLRELIRLKDELIAKSNTPPMYLQADIEAFDIRELTPKFDVILLEPPLEEYYRETGITANEKCWTWDDIMKLEIDEIAAPRSFIFLWCGSGEGLDLGRVCLRKWGYRRCEDICWIKTNKNNPGKTKTLDPKAVFQRTKEHCLMGIKGTVKRSTDGDFIHANVDIDLIITEEPEIGNIEKPVEIFHIIEHFCLGRRRLHLFGRDSTIRPGWLTVGPTLTNSNYNAETYASYFSAPNSYLTGCTEEIERLRPKS
Vector:pFBOH-MHL

Growth

Medium:
Antibiotics:
Procedure:Sf9 cells were infected with virus and incubated at 27°C for 48-72 hours until cell viability drops to 70-80%.

Extraction

Buffers
Procedure
Cells were harvested by centrifugation at 4,000 rpm at 4°C for 15 minutes. After removing the medium, cells were washed with cold 1X PBS. PBS was removed after centrifugation and the pellet was resuspended in suspension buffer (20 mM Tris, pH 8.0, 250 mM NaCl, 5% glycerol, 2 mM β-mercaptoethanol, 0.6 % NP-40, 1 X protease inhibitor cocktail (Roche), 5 mM imidazole The cell pellets were frozen in liquid nitrogen and stored at -80°C.

Purification

Buffers
Procedure
For purification the cell paste was thawed and 3000 U of benzonase (Novagen) were added. Cells were lyzed by brief sonication. The clarified lysate was loaded onto a 2 mL TALON column (Clonetech). The column was washed with 50 column volumes of 20 mM Tris-HCl buffer, pH 8.0, containing 250 mM NaCl, 5% cglycerol and 5 mM imidazole, and the protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 5% glycerol, 250 mM imidazole). The eluted protein was loaded on a Superdex200 column (GE Healthcare), equilibrated with 20 mM HEPES buffer, pH 7.4, and 250 mM NaCl. Pooled fractions containing METTL3/METLTTL14 were further purified by another round of gel filtration after auto proteolysis.

Concentration:12.6 mg/ml
Ligand
MassSpec:Measured mass are 25413.84 Da and 33914.49 Da.
Crystallization:Purified METTL3/METTL14 (12 mg/mL) was complexed with S-adenosyl-L-methionine (SAM, Sigma) at 1:10 molar ratio of protein:SAM and crystallized using hanging drop vapor diffusion method at 20 °C by mixing 2 µl of the protein solution with 2 µl of the reservoir solution containing 20% PEG4000, 0.2 M MgCl2, 0.1 M Tris, pH 8.5.
NMR Spectroscopy:
Data Collection:
Data Processing: