Human methyltransferase like 1 in complex with SAM

Target ID:

METTL1

Aliases:

C12orf1FLJ95748TRM8YDL201w

Deposition Date:

Sunday 16th March 2008

Families:

Transferases

Related Diseases:

Metabolism

Structure type:

protein-ligand

Download Coordinates:

3CKK

Authors:

Dong A, Zeng H, Dobrovetsky E, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Min J, Plotnikov AN, Wu H

Site:

Toronto

ICM Datapack:

ICM Datapack

3CKK

tabs

Structure Details

The methylation of a wide variety of biologically active molecules is a common theme in biology. The functional roles of methylation are wide ranging and include biosynthesis, metabolism, detoxification, signal transduction, protein sorting and repair, nucleic acid processing, gene silencing and imprinting. The majority of methylation reactions are carried out by the S-adenosylmethionine (AdoMet)-dependent methyl transferases.

Functional tRNAs carry a number of chemically modified nucleosides that are formed enzymatically after transcription, during the tRNA maturation process. These modifications are catalyzed by various enzymes including methyltransferase-like 1 (METTL1) protein. METTL1 protein is highly conserved in prokaryotes and eukaryotes. It catalyzes the formation of N(7)-methylguanine at position 46 (m7G46) in tRNA. 7-methylguanosine (m7G) modification of tRNA occurs widely in eukaryotes and bacteria. This modification is nearly always found at position 46, and is one of the few modifications that confer a positive charge to the base. Loss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. Human METTL1 contains an S-adenosylmethionine binding motif and functions in complex with WDR4 protein. METTL1 protein has been found to be regulated by phosphorylation and growth factor. This finding indicates that the m7G methylation complex is involved in a wide variety of cellular processes.

We have sovled the crystal structure of human METTL1 protein in complex with S-adenosyl-L-methionine at 1.55 Å resolution.

Materials and Methods

METTL1

PDB:3CKK

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_005362
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQ*G
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).

Construct

Prelude:
Sequence:gDHTLRYPVKPEEMDWSELYPEFFAPLTQNQSHDDPKDKKEKRAQAQVEFADIGCGYGGLLVELSPLFPDTLILGLEIRVKVSDYVQDRIRALRAAPAGGFQNIACLRSNAMKHLPNFFYKGQLTKMFFLFPDPHFKRTKHKWRIISPTLLAEYAYVLRVGGLVYTITDVLELHDWMCTHFEEHPLFERVPLEDLSEDPVVGHLGTSTEEGKKVLRNGGKNFPAIFRRIQDPVLQ
Vector:pET28a-LIC

Growth

Medium:TB
Antibiotics:
Procedure:METTL1 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 37 degC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM, and incubated overnight at 15 degC.

Purification

Procedure
Column 1: 5 ml HiTrap column (Amersham Biosciences)
Column 2: Source 30S column (10x10) (Amersham Biosciences)

The crude extract was cleared by centrifugation. The lysate was loaded onto 5 ml HiTrap column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of wash buffer, and the protein was eluted with elution buffer. The fractions containing METTL1 protein was pooled and dialyzed against 20 mM HEPES buffer, pH 7.4, and 250 mM NaCl, in the presence of TEV, overnight at 4 degC. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30S column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM PIPES, pH 6.5, and eluted with linear gradient of NaCl up to 500 mM concentration (20CV). Purification yield was 7 mg of the protein per 1L of culture.

Extraction

Procedure
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 with protease inhibitor (1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:28.5 mg/ml
Ligand
MassSpec:The expected mass for METTL1 is 26997.03 Da, measured mass is 26997.00 Da.
Crystallization:Purified METTL1 protein was complexed with S-adenosyl-L-methionine (SAM) (Sigma) at 1:10 molar ratio of protein:SAM and crystallized using sitting drop vapor diffusion method at 20°C by mixing 1 microL of the protein solution with 1 microL of the reservoir solution containing 2.0 M ammonium sulfate, 2% PEG400, 0.1 M Na-HEPES, pH 7.5.
NMR Spectroscopy:
Data Collection:
Data Processing: