Human methyltransferase like 21C, catalytic domain, in complex with S-adenosylhomocysteine

Target ID:

METTL21C

Deposition Date:

Thursday 19th September 2013

Families:

Methyltransferases

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

4MTL

Authors:

Hong BS, Tempel W, Dong A, Li Y, Arrowsmith CH, Bountra C, Edwards AM, Brown PJ

Site:

Toronto

4MTL

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 METTL21C, 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

METTL21C

PDB:4MTL

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:SGC cDNA collection CM23-C12
Entry Clone Source:MGC clone collection
SGC Clone Accession:PBC002-E06
Tag:N-terminal His6-tag, removed before crystallization
Host:BL21-V2R-pRARE2

Construct

Prelude:METTL21C:G22-D264
Sequence:gGGWLEAEKKGAPQKDSTGGVLEESNKIEPSLHSLQKFVPTDYASYTQEHYRFAGKEIVIQESIESYGAVVWPGAMALCQYLEEHAEELNFQDAKILEIGAGPGLVSIVASILGAQVTATDLPDVLGNLQYNLLKNTLQCTAHLPEVKELVWGEDLDKNFPKSAFYYDYVLASDVVYHHYFLDKLLTTMVYLSQPGTVLLWANKFRFSTDYEFLDKFKQVFDTTLLAEYPESSVKLFKGILKWD
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 anion-exchange chromatography using a HiTrap Q column (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:27 mg/mL
Ligand
S-Adenosyl-L-homocysteine (SAH)MassSpec:cut version, expected is 27464.1 Da and the measured value was 27464.5 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 SGC screen condition E08 (30% PEG3350, 0.2M NaCl, 0.1M HEPES pH7.5) 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.