Human Lethal(3)malignant brain tumor-like 2 protein in complex with H4K20me

Target ID:

L3MBTL2

Aliases:

H-l(3)mbt-lL3MBT

Deposition Date:

Saturday 31st May 2008

Families:

Methyl-lysine readers

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

Superceded by 3F70

Authors:

Guo Y, Qi C, Allali-Hassani A, Pan P, Zhu H, Dong A, Mackenzie F, Crombet L, Loppnau P, Kozieradzki I, Vedadi M, Edwards AM, Weigelt J, Bountra C, Arrowsmith CH, Bochkarev A, Read R, Min J.

Site:

Toronto

3DBB

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Structure Details

MBT repeat proteins are a family of proteins involved in transcriptional repression although their exact biological function is unclear. The MBT (malignant brain tumor) domain shows structural similarity with the Chromo-, Tudor and PWWP-domains, which are jointly referred to as "Royal Family" domain. Considering Tudor domain and Chromodomain have been proved to be histone binding domains, it is predicted that MBT domain can also bind modified histones. Recently, dSfMBT protein in PhoRC complex was shown to preferentially binds mono- or di-methylated H3K9 and H4K20 peptides. PRE-tethered PhoRC selectively interacts with methylated histones in the chromatin flanking PREs to maintain a Polycomb-repressed chromatin state.(Klymenko et al, Genes & Dev, 2006).

L3MBTL2, like L3MBTL1, is also implicated in transcriptional repression. L3MBTL2 contains 4 MBT repeats. In order to elucidate the molecular mechanisms of the MBT domain in histone binding and transcriptional repression, we carried out structural study of this protein. The crystal structure of L3MBTL2 protein in complex with H4K20me reveals that L3MBTL2 has distorted rhombus architecture and the details of substrate binding.

Materials and Methods

L3MBTL2

PDB:3DBB

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_113676
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:mhhhhhhssgrenlyfqgTGQDALVLGFDWGKFLKDHSYKAAPVSCFKHVPLYDQWEDVMKGMKVEVLNSDAVLPSRVYWIASVIQTAGYRVLLRYEGFENDASHDFWCNLGTVDVHPIGWCAINSKILVPPRTIHAKFTDWKGYLMKRLVGSRTLPVDFHIKMVESMKYPFRQGMRLEVVDKSQVSRTRMAVVDTVIGGRLRLLYEDGDSDDDFWCHMWSPLIHPVGWSRRVGHGIKMSERRSDMAHHPTFRKIYCDAVPYLFKKVRAVYTEGGWFEEGMKLEAIDPLNLGNICVATVCKVLLDGYLMICVDGGPSTDGLDWFCYHASSHAIFPATFCQKNDIELTPPKGYEAQTFNWENYLEKTKSKAAPSRLFNMDCPNHGFKVGMKLEAVDLMEPRLICVATVKRVVHRLLSIHFDGWDSEYDQWVDCESPDIYPVGWCELTGYQLQPPVAAEPATPLKAKEATKKKKKQ
Vector:pET28-MHL

Growth

Medium:TB
Antibiotics:
Procedure:L3MBTL2 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: Affinity purification, Ni-NTA
Column 2: Gel filtration, HiLoad 26/60 Superdex 200

The supernatant was incubated with 6mL of 50% slurry Ni-NTA beads by rocking. After 1 hour incubation at 4 ºC, the beads were washed with 50 mL of lysis buffer. The protein was eluted using ~20mL EB.

The eluent from Ni column was loaded onto the gel filtration column in GF buffer at 2.5 mL/min, fraction size 4mL. The fractions containing protein were identified on a SDS-PAGE gel.

Extraction

Procedure
Cells were harvested by centrifugation and pellets were stored in -80 ºC. Prior to purification, the cell pellet was resuspended in lysis buffer. Cells were disrupted by sonication (10 minutes twice ) and samples were centrifuged for 60 min at 70000 g. The soluble fraction was subjected to further purification by affinity and size exclusion chromatography.
Concentration:10.5 mg/ml
Ligand
MassSpec:
Crystallization:100mM NaAc 5.3, 100mM NaAC, 15% PEG 3350
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Klymenko T, Papp B, Fischle W, Kocher T, Schelder M, Fritsch C, Wild B, Wilm M, Muller J. (2006) A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities. Genes Dev. 20(9):1110-22.
Trojer P, Li G, Sims RJ, Vaquero A, Kalakonda N, Boccuni P, Lee D, Erdjument-Bromage H, Tempst P, Nimer SD, Wang YH, Reinberg D (2007) L3MBTL1, a histone-methylation-dependent chromatin lock. Cell. 129(5):915-28.