Human L3MBTL protein in complex with H4K20Me2

Target ID:

L3MBTL1

Aliases:

dJ138B7.3DKFZp586P1522FLJ41181H-L(3)MBTKIAA0681L3MBTL1

Deposition Date:

Wednesday 17th October 2007

Families:

Methyl-lysine readers

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

2RJF

Authors:

Allali-Hassani, A., Liu, Y., Herzanych, N., Ouyang, H., Mackenzie, F., Crombet, L., Loppnau, P., Kozieradzki, I., Vedadi, M., Weigelt, J., Sundstrom, M., Arrowsmith, C.H., Edwards, A.M., Bochkarev, A., Min, J.R.

Site:

Toronto

2RJF

tabs

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).

L3MBTL1, the human homolog of the Drosophila tumor suppressor protein D-l(3)mbt, was previously shown to be a repressor of transcription and its mode of action was independent of HDACs. It was reported that L3MBTL1 is involved in chromatin condensation through H1K26 and H4K20 methylation in Mammals (Trojer, Cell, 2007). In order to elucidate the molecular mechanisms of the MBT domain in histone binding,, we carried out structural study of this protein. the crystal structure of L3MBTL1 in complex with H4K20me2 reveals how MBT domain selectively binds mono- or di-methylated lysine histones, not unmodified or trimethylated lysine histones.

We have solved the crystal structure of human L3MBTL1 bound to H4K20Me2 peptide, orthorhombic form I.

Materials and Methods

L3MBTL

PDB:2RJF

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_056293
Entry Clone Source:
SGC Clone Accession:L3MBTL1_3:A3-APC053
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgrenlyfq*g
Host:E Coli BL21(DE3) RIL CodonPlus

Construct

Prelude:
Sequence:GEKKECWSWESYLEEQKAITAPVSLFQDSQAVTHNKNGFKLGMKLEGIDPQHPSMYFILTVAEVCGYRLRLHFDGYSECHDFWVNANSPDIHPAGWFEKTGHKLQPPKGYKEEEFSWSQYLRSTRAQAAPKHLFVSQSHSPPPLGFQVGMKLEAVDRMNPSLVCVASVTDVVDSRFLVHFDNWDDTYDYWCDPSSPYIHPVGWCQKQGKPLTPPQDYPDPDNFCWEKYLEETGASAVPTWAFKVRPPHSFLVNMKLEAVDRRNPALIRVASVEDVEDHRIKIHFDGWSHGYDFWIDADHPDIHPAGWCSKTGHPLQPPLGPRE
Vector:p28a-MHL

Growth

Medium:TB
Antibiotics:
Procedure:A glycerol stock was used to innoclate 20 mL LB media containing 50 µg/mL kanamycin. The culture was grown overnight at 37ºC with shaking. The next day this starter culture was used to innoculate 2L of TB medium which contained 50 μg/mL kanamycin. The culture was grown in LEX at 37ºC to OD600 ~1.1 and was induced with the addition of 0.5 mM IPTG. The temperature was reduced to 18ºC and the culture was incubated for a further 16 hours before harvesting the cells.

Purification

Procedure
Column 1: Affinity purification, open Ni-NTA columnProcedure: The supernatant was incubated with 6mL of 50% slurry Ni-NTA beads by rocking. After 1 hour incubation at 4ºC, the beads were washed with 50 mL of lysis buffer. The protein was eluted using ~20mL EB. Column 2: Gel filtration, HiLoad 26/60 Superdex 200 Prep GradeProcedure: 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 -80ºC. 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:20mg/ml
Ligand
MassSpec:
Crystallization:100mM NaAc 4.6, 100mM NaAC, 5% PEG 4K
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