Human MBT domain containing 1

Target ID:

MBTD1

Deposition Date:

Sunday 30th November 2008

Families:

Methyl-lysine readers

Related Diseases:

Epigenetics

Structure type:

apo

Download Coordinates:

3FEO

Authors:

Amaya MF, Eryilmaz J, Kozieradzki I, Edwards AM, Arrowsmith CH, Weigelt J, Bountra C, Bochkarev A, Min J

Site:

Toronto

3FEO

tabs

Structure Details

Malignant Brain Tumour domain-containing protein 1 (MBTD1) belongs to the Polycomb group (PcG) of proteins that function as important developmental regulators in large protein complexes. The malignant-brain-tumor (MBT) repeat is a structural motif of ~100 amino acids, originally identified in the Drosophila tumor-suppressor protein L(3)mbt[1]. Mutations of the D-l(3)mbt gene cause malignant transformation in the larval brain. Based on the sequence analysis, MBT repeat shows structural similarity with the Chromo-, Tudor and PWWP-domains, which together are referred to as "Royal Family" domain[2]. It has been implicated that the structural similarity between these domains reflect a common function in binding methylated lysine residues[3,4].

We have determined the structure of MBTD1 (residues 130-566) at 2.5Å resolution. The overall structure reveals two MBTD1 molecules in the crystal lattice. Each MBTD1 molecule contains four MBT repeats with similar structure to other MBT domains that bind lower methylated lysine histones with no apparent sequence specificity. Fluorescence polarization experiments confirmed that the fourth domain of MBTD1 preferentially binds histone peptides at mono- and di-methyllysine residues but unexpectedly they show that MBTD1 binds those peptides with sequence specificity for H4K20 histone peptide.

Materials and Methods

MBTD1

PDB:3FEO

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_060113.1
Entry Clone Source:
SGC Clone Accession:MBTD1_5:E5-JMC013
Tag:N-terminal hexahistidine tag
Host:E. Coli BL21 (DE3)-V2R-pRARE2

Construct

Prelude:
Sequence:AKTKAAVSMEGFSWGNYINSNSFIAAPVTCFKHAPMGTCWGDISENVRVEVPNTDCSLPTKVFWIAGIVKLAGYNALLRYEGFENDSGLDFWCNICGSDIHPVGWCAASGKPLVPPRTIQHKYTNWKAFLVKRLTGAKTLPPDFSQKVSESMQYPFKPCMRVEVVDKRHLCRTRVAVVESVIGGRLRLVYEESEDRTDDFWCHMHSPLIHHIGWSRSIGHRFKRSDITKKQDGHFDTPPHLFAKVKEVDQSGEWFKEGMKLEAIDPLNLSTICVATIRKVLADGFLMIGIDGSEAADGSDWFCYHATSPSIFPVGFCEINMIELTPPRGYTKLPFKWFDYLRETGSIAAPVKLFNKDVPNHGFRVGMKLEAVDLMEPRLICVATVTRIIHRLLRIHFDGWEEEYDQWVDCESPDLYPVGWCQLTGYQLQPPASQSSR
Vector:p28a-MHL

Growth

Medium:TB
Antibiotics:
Procedure:A glycerol stock was used to inoculate 20 mL LB media containing 50 µg/mL kanamycin. The culture was grown overnight at 37°C with shaking. The next day this starter culture was used to inoculate 2L of TB medium which contained 50 μg/mL kanamycin. The culture was grown in LEX at 37°C to OD600 ~3.0 and was induced with the addition of 0.5 mM IPTG. The temperature was reduced to 14°C and the culture was incubated for a further 16 hours before harvesting the cells.

Purification

Procedure
Column 1: Affinity purification: open Ni-NTA Superflow column.
Column 2: Gel filtration: HiLoad 26/60 Superdex 75 Prep
Column 3: Ion exchange: HiTrapQ 5ml.

The supernatant was incubated with 6ml of 50% slurry Ni-NTA beads on ice. After 1 hour incubation the beads were washed with 100ml of lysis buffer. The protein was eluted using 15mL of EB.

The eluent from Ni column was loaded onto the gel filtration column in GF buffer at 1ml/min, fraction size 2mL.

The fractions containing protein were loaded onto the HiTrapQ column containing bufferA at 3ml/min and eluted with gradient of bufferB. 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. The cells were homogenised and sonicated on ice with 10second pulses and 30 second rest intervals. The output level was 8.5 for 6 mins. The sample was centrifuge at 16,000rpm for 1 hour. The supernatant was purified by affinity, size exclusion and ion exchange.
Concentration:10mg/ml.
Ligand
MassSpec:
Crystallization:crystals were obtained by macroseeding in solution containing 20% PEG 3350, 0.2M CaOAc
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Wismar, J., Loffler, T., Habtemichael, N., Vef, O., Geissen, M., Zirwes, R., Altmeyer, W., Sass, H., and Gateff, E. The Drosophila melanogaster tumor suppressor gene lethal(3)malignant brain tumor encodes a proline-rich protein with a novel zinc finger. Mech. Dev. 53, 141-154 (1995)
Maurer-Stroh, S. et al. The Tudor domain 'Royal Family': Tudor, plant Agenet, Chromo, PWWP and MBT domains. Trends Biochem. Sci. 28, 69-74 (2003)
Kim, J., Daniel, J., Espejo, A., Lake, A., Krishna, M., Xia, L., Zhang, Y., and Bedford, M.T. Tudour, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep. 4, 397-403 (2006).
Klymenko, T., Papp, B., Fischle, W., Kocher, T., Schelder, M., Fritsch, C., Wild, B., Wilm, M., and Muller, J. A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities. Genes Dev. 20, 1110-1122 (2006).