Human methyltransferase like 18, methyltransferase domain, in complex with S-adenosylmethionine

Target ID:

METTL18

Deposition Date:

Tuesday 30th September 2014

Families:

Methyltransferases

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

4RFQ

Authors:

Tempel W, Ravichandran M, Li Y, Walker JR, Bountra C, Arrowsmith CH, Edwards AM, Brown PJ, Hong BS

Site:

Toronto

4RFQ

tabs

Structure Details

Family 16 methyltransferases (InterPro: IPR019410; Pfam:PF10294) contains ten human members which are classified as a 'Class I methyltransferase superfamily'. Recent studies have reported that some of this family proteins specifically methylate lysine residues on chaperones, thereby modulating biological functions of large chaperone molecules which target other cellular proteins. The overexpression or methylation activity of some of this family members is known to be significantly related not only to a number of metastatic tumours , but also to human neurodegenerative disorders, indicating their dynamic involvement in human diseases, although the mechanism underlying remain elusive so far. Crystal structure of METTL18, one of Family 16 methyltransferases, shows a single SAM-dependent methyltransferase domain adopting a canonical Rossman-like fold with a central seven-beta stranded beta-sheet flanked by five alpha helices. Our model was also determined in complex with SAH and provides the three-dimensional clues about the mode of access and binding of substrate to the active site of the protein.

Materials and Methods

METTL18

PDB:4RFQ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:SGC cDNA collection AT6-C11
Entry Clone Source:MGC clone collection
SGC Clone Accession:PBC004-E03
Tag:N-terminal His6-tag, removed before crystallization
Host:BL21-V2R-pRARE2

Construct

Prelude:METTL18 :H63-G372
Sequence:gHKSMENAAPSQDTDSPLSAASSSRNLEPHGKQPSLRAAKEHAMPKDLKKMLENKVIETLPGFQHVKLSVVKTILLKENFPYEGGLKIWECTFDLLAYFTKAKVKFAGKKVLDLGCGSGLLGITAFKGGSKEIHFQDYNSMVIDEVTLPNVVANSTLEDEENDVNEPDVKRCRKPKVTQLYKCRFFSGEWSEFCKLVLSSEKLFVKYDLILTSETIYNPDYYSNLHQTFLRLLSKNGRVLLASKAHYFGVGGGVHLFQKFVEERDVFKTRILKIIDEGLKRFIIEITFKFPG
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:LEX Bubbling. The target protein was expressed in E. coli by inoculating each 50 mL of overnight culture grown in Luria-Bertani medium into a 2 L of Terrific Broth medium in the presence of 50 mg/mL kanamycin and 30 mg/mL chloramphenicol at 37 °C. When OD600 reached ~3.0, the temperature of the medium was lowered to 18 °C and the culture was induced with 0.5 mM IPTG. The cells were allowed to grow overnight before harvested and then flash frozen in liquid nitrogen and stored at -80 degree until purified

Purification

Procedure
The cell lysate was centrifuged to remove insoluble material, and the supernatant was loaded onto a DEAE-cellulose (DE52, Whatman, MA, USA) anion-exchange resin followed by a nickel-NTA agarose column (Qiagen, MD, USA). Bound proteins were eluted with Elution buffer. The eluted sample was mixed with TEV in a 1:4 molar ratio (TEV to protein) and then dialyzed and subjected to cation-exchange chromatography using a HiTrap SPcolumn (GE Healthcare, NJ, USA) previously equilibrated with the Ion Exchange A buffer. Protein was eluted with a linear gradient of 0-500 mM NaCl using Ion Exchange B buffer, and further purified by size exclusion chromatography on a Superdex 75 16/60 column (GE Healthcare, NJ, USA). The peak fractions containing target protein were pooled, concentrated using amicon centrifugal filter and stored at -80 °C before crystallization. The purity and molecular weight of the final protein sample were confirmed by SDS-PAGE and LC-MS, respectively.

Extraction

Procedure
Frozen cells were thawed and suspended in 150 mL extraction buffer and lysed using a Microfluidizer at 16,000 psi. The lysate was then clarified by centrifugation at ~38000 x g (15,500 rpms) for 1 hour.
Concentration:37.5 mg/mL
Ligand
S-Adenosyl-L-homocysteine (SAH)MassSpec:cut version, expected is 33021.1 Da and the measured value was 33022.5 Da
Crystallization:Protein sample was incubated with SAH (final concentration of 5mM) for overnight and set up using SGC and Red Wing (RW) screen conditions at room temperature. Crystals were initially found in RW screen condition G01 (2.0M (NH4)2SO4, 0.2 K/Na tartrate, 0.1M Na-Citrate pH5.6, 5mM Taurine) and used for structure determination.

Mineral oil : Paratone oil = 50 : 50
NMR Spectroscopy:
Data Collection:
Data Processing:

References

1. Kernstock, S., et al. (2012). "Lysine methylation of VCP by a member of a novel human protein methyltransferase family." Nat Commun 3: 1038.

2. Cloutier, P., et al. (2013). "A newly uncovered group of distantly related lysine methyltransferases preferentially interact with molecular chaperones to regulate their activity." PLoS Genet 9(1): e1003210.