Human protein arginine methyltransferase 6 in complex with an allosteric inhibitor

Target ID:

HRMT1L6

Deposition Date:

Tuesday 24th June 2014

Families:

Methyltransferases

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

4QPP

Authors:

Dong A, Zeng H, Smil D, He H, Eram M, Bountra C, Arrowsmith CH, Edwards AM, Vedadi M, Brown PJ, Wu H

Site:

Toronto

4QPP

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) and inhibitor DS-421 (2-{4-[3-chloro-2-(2-methoxyphenyl)-1H-indol-5-yl]piperidin-1-yl}-N-methylethanamine) at 2.52 Å resolution.

Materials and Methods

PRMT6

PDB:4QPP

Revision

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

Construct

Prelude:
Sequence:gMSQPKKRKLESGGGGEGGEGTEEEDGAEREAALERPRRTKRERDQLYYECYSDVSVHEEMIADRVRTDAYRLGILRNWAALRGKTVLDVGAGTGILSIFCAQAGARRVYAVEASAIWQQAREVVRFNGLEDRVHVLPGPVETVELPEQVDAIVSEWMGYGLLHESMLSSVLHARTKWLKEGGLLLPASAELFIAPISDQMLEWRLGFWSQVKQHYGVDMSCLEGFATRCLMGHSEIVVQGLSGEDVLARPQRFAQLELSRAGLEQELEAGVGGRFRCSCYGSAPMHGFAIWFQVTFPGGESEKPLVLSTSPFHPATHWKQALLYLNEPVQVEQDTDVSGEITLLPSRDNPRRLRVLLRYKVGDQEEKTKDFAMED
Vector:pFBOH-MHL

Growth

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

Purification

Procedure
For purification the cell paste was thawed and resuspended in lysis buffer containing 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazol, 2 mM β-mercaptoethanol, 5% glycerol, 0.6% NP-40, protease inhibitor cocktail (Roche), 3000 U of benzonase (Novagen). 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 500 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 Tris-HCl buffer, pH 8.0, and 150 mM NaCl. Pooled fractions containing HRMT1L6 were subjected to TEV treatment to remove His-tag. The protein was further purified to homogeneity by ion-exchange chromatography.

Extraction

Procedure
Cells were harvested by centrifugation at 5,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80˚C.
Concentration:16 mg/ml - Enzymatic treatment: TEV
Ligand
MassSpec:expected mass is 42022.6 Da, measured mass is 42132.48 Da.
Crystallization:Purified HRMT1L6 (4.9 mg/ml) was complexed with S-adenosyl-L-homocysteine (SAH, Sigma) and DS-421 at 1:5 molar ratio of protein:compound and crystallized using the hanging drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution with 1 µl of the reservoir solution containing 2.0 M sodium formade, 0.1 M sodium acetate, pH 4.6.
NMR Spectroscopy:
Data Collection:
Data Processing: