Human protein arginine methyltransferase 6, methyltransferase domain

Target ID:

HRMT1L6

Deposition Date:

Friday 28th September 2012

Families:

Methyltransferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

4HC4

Authors:

Dong A, Zeng H, He H, El Bakkouri M, Bountra C, Arrowsmith CH, Edwards AM, Brown PJ, Wu H

Site:

Toronto

4HC4

tabs

Structure Details

Protein arginine methylation is a common posttranslational modification process in
eukayotes. There are two major types of protein arginine methyltransferases (PRMTs)
that transfer the methyl group from S-adenosyl-L-methionine (SAM) to the guanidino
group of arginine in protein substrates. Both groups of PRMTs catalyze the formation
of monomethyl-arginine (MMA) but differ in the dimethylation products: type I
PRMTs form asymmetric dimethylarginine (aDMA); type II PRMTs form symmetric
dimethylarginine (sDMA). The posttranslational methylation of arginine residues has
been shown to modify protein function by regulating protein-protein interactions. It is
also involved in a number of processes such as DNA repair, RNA transcription, signal
transduction, protein compartmentalization.

HRMT1L6 is a member of the type I PRMT family. It can catalyze the formation of
monomethylarginine and asymmetrical dimethylarginine, with a strong preference for the
formation of aDMA. HRMT1L6 specifically mediates the asymmetrical dimethylation of
histone H3R2. H3R2me2a is mutually exclusive with methylation of H3K4, which is a
mark for transcriptional activation, it therefore represents a specific tag for transcriptional
repression. Besides this, HRMT1L6 also has a wide range of substrates including histone
H2A (H2AR3), H4 (H4R3), DNA polymerase beta (POLB), HMGA1 and various HIV-
1 proteins. It thereby plays a role in different processes such as DNA repair, innate
immunity against HIV-1. We have solved the crystal structure of human HRMT1L6 in
complex with S-adenosyl-L-homocysteine (SAH) at 1.97 Å resolution.

Materials and Methods

PRMT6

PDB:4HC4

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:227908866
Entry Clone Source:MGC
SGC Clone Accession:HRMT1L6:PBC001-A12:C213642
Tag:N-terminal: His-tag with integrated TEV protease site:MHHHHHHSSGRENLYFQ*G
Host:Sf9

Construct

Prelude:
Sequence:gMSQPKKRKLESGGGGEGGEGTEEEDGAEREAALERPRRTKRERDQLYYECYSDVSVHEEMIADRVRTDAYRLGILRNWAALRGKTVLDVGAGTGILSIFCAQAGARRVYAVEASAIWQQAREVVRFNGLEDRVHVLPGPVETVELPEQVDAIVSEWMGYGLLHESMLSSVLHARTKWLKEGGLLLPASAELFIAPISDQMLEWRLGFWSQVKQHYGVDMSCLEGFATRCLMGHSEIVVQGLSGEDVLARPQRFAQLELSRAGLEQELEAGVGGRFRCSCYGSAPMHGFAIWFQVTFPGGESEKPLVLSTSPFHPATHWKQALLYLNEPVQVEQDTDVSGEITLLPSRDNPRRLRVLLRYKVGDQEEKTKDFAMED
Vector:pFBOH-MHL

Growth

Medium:HRMT1L6 was expressed in Sf9 cells
Antibiotics:
Procedure:

Purification

Procedure
For purification the cell paste was thawed and resuspended inlysis buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazol, 2 mMß-mercaptoethanol, 5% glycerol, 0.6% NP-40, protease inhibitor cocktail (Roche), 3000U of benzonase (Novagen). Cells were lyzed by brief sonication. The clarified lysatewas loaded onto a 2 mL TALON column (Clonetech). The column was washed with50 column volumes of 20 mM Tris-HCl buffer, pH 8.0, containing 500 mM NaCl, 5%cglycerol and 5 mM imidazole, and the protein was eluted with elution buffer (20 mMTris-HCl, pH 8.0, 250 mM NaCl, 5% glycerol, 250 mM imidazole). The eluted proteinwas loaded on a Superdex200 column (GE Healthcare), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 150 mM NaCl. Pooled fractions containing HRMT1L6 weresubjected to TEV treatment to remove His-tag. The protein was further purified tohomogeneity by ion-exchange chromatography.

Enzymatic treatment: TEV

Extraction

Procedure
Cells were harvested by centrifugation at 5,000rpm. The cell pellets were frozen in liquid nitrogen and stored at -80°C.
Concentration:16 mg/ml
Ligand
MassSpec:expected mass is 42022.6 Da, measured mass is 42132.48Da.
Crystallization:Purified HRMT1L6 (4.9 mg/ml) was complexed with (SAH, Sigma) at1:5 molar ratio of protein:SAH and crystallized using the hanging drop vapor diffusionmethod at 20 °C by mixing 1 µl of the protein solution with 1 µl of the reservoir solutioncontaining 15% PEG3350, 0.1 M succinate acid, pH 7.0.
NMR Spectroscopy:
Data Collection:
Data Processing: