Human chromobox homolog 3 in complex with a tri-methylated lysine peptide

Target ID:

CBX3

Deposition Date:

Monday 30th June 2008

Families:

Methyl-lysine readers

Related Diseases:

CancerChromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

3DM1

Authors:

Amaya MF, Ravichandran M, Loppnau P, Kozieradzki I, Edwards AM, Arrowsmith CH, Wikstrom M, Bountra C, Bochkarev A, Min J, Ouyang H

Site:

Toronto

ICM Datapack:

ICM Datapack

3DM1

tabs

Structure Details

Chromodomain-containing proteins are a family of effector proteins, regulating various downstream activities. For example, position effect variegation (PEV) is regulated by HP1[1] and homeotic gene silencing is regulated by polycomb[2], whose chromdomain recognizes histone H3 methylated at K9 and K27, respectively[3-6]. Human CHD1 binds directly and selectively to histone H3 methylated at lysine 4 via its tandem chromodomains, which is associated with transcriptional activity[7]. The chromodomain of Eaf3 interacts with methylated H3-K36, linking histone deacetylation to Pol II elongation[8]. In the human genome, there are over 30 chromodomain proteins. Despite a high degree of conservation, the chromodomains display significant differences in binding preferences[9]. Besides, the biological functions for most of them are not clear.

HP1 and PC proteins have been extensively studied functionally, with strong evidence that HP1 preferentially binds methylated H3K9, while PC selectively binds H3K27me3. So far just 2 complex structures have been solved for each, which illustrated the structural mechanism for this binding selectivity. In order to better understand such selectivity family-wide, we aim to solve all human chromodomain proteins, and thoroughly analyze the binding preference of this protein family. CBX3 is one of them, and is also a human homolog of HP1.

We solved the structure of CBX3 in complex with a G9a peptide. The structure shows that, like HP1-H3K9me3, the modified lysine is accommodated and recognized by an aromatic cage. The G9a peptide is bound in a cleft formed on one face of the chromodomain of CBX3.

Materials and Methods

CBX3

PDB:3DM1

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_009207
Entry Clone Source:MGC AT2-F11 (BC000954)
SGC Clone Accession:
Tag:N-terminal tag: MHHHHHHSSGRENLYFQG
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgEFVVEKVLDRRVVNGKVEYFLKWKGFTDADNTWEPEENLDCPELIEAFLNSQK
Vector:pET28a-mhl

Growth

Medium:TB
Antibiotics:
Procedure:A 250 ml flask containing LB (Sigma L7658) supplemented with 50 ug/ ml kanamycin (BioShop Canada KAN 201) was inoculated from a glycerol stock of the bacteria. The flask was shaken overnight (16 hours) at 250 rpm at 37 degC. Using the Lex system, a 2L bottle (VWR 89000-242) containing 1800 ml of TB (Sigma T0918) supplemented with 1.5% glycerol, 50 ug/ ml kanamycin and 600 ul antifoam 204 (Sigma A-8311) was inoculated with 50 ml overnight LB culture, and incubated at 37 degC. The temperature of the media was reduced to 15 degC one hour prior to induction and induced at OD(600) = 6 with 100 uM isopropyl-thio-β-D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15 degC, and cell pellets collected by centrifugation and frozen at -80 degC.

Purification

Procedure
IMAC: Unclarified lysate was mixed with 2-3 mL of Ni-NTA superflow Resin (Qiagen) per 40 mL lysate. The mixture was incubated with mixing for at least 45 minutes at 4oC. The mixture was than loaded onto an empty comlum (BioRad) and washed with 100 ml wash buffer. Samples were eluted from the resin by exposure to 2-3 column volumes (approx. 10-15 mL) of elution buffer. Concentration of eluted protein was estimated by OD280. TEV protease was added at 1 unit per mg of eluted protein and dialyzes against gel filtration buffer overnight to remove His-tag.

Gel filtration chromatography: An XK 26x65 column (GE Healthcare) packed with HighLoad Superdex 75 resin (GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTA explorer (GE Healthcare) at a flow rate of 2.5mL/min. The dialyzed sample from the IMAC step (approx. 15 mL) was loaded onto the column at 1.5 mL/min, and 2mL fractions were collected into 96-well plates (VWR 40002-012) using peak fractionation protocols). Fractions observed by a UV absorption chromatogram to contain the protein were pooled.

Extraction

Procedure
Frozen cell pellet contained in bags (Beckman 369256) obtained from 2L of culture were thawed by soaking in warm water. Each cell pellet was resuspended in 25-40 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 10 minutes total sonication time per pellet.
Concentration:Purified proteins were concentrated using 15 mL concentrators with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration around 15 mg/mL.
Ligand
MassSpec:
Crystallization:CBX 3 (26mg/ml) and the trimethylated lysine peptide were mixed in the ratio 1:1.2 and incubated overnight at 4 degC and the hanging drop was setup against the well solution of 40% PEG 550MME at 298 K at1:1 ratio. Crystals appeared after 4 days and were flash frozen in liquid nitrogen and the data was collected at 2.1A.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Grewal, S.I. & Jia, S. Heterochromatin revisited. Nat Rev Genet 8, 35-46 (2007).
Cao, R. & Zhang, Y. The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3. Curr Opin Genet Dev 14, 155-64 (2004).
Fischle, W. et al. Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev 17, 1870-81 (2003).
Jacobs, S.A. & Khorasanizadeh, S. Structure of HP1 chromodomain bound to a lysine 9-methylated histone H3 tail. Science 295, 2080-3 (2002).
Min, J., Zhang, Y. & Xu, R.M. Structural basis for specific binding of Polycomb chromodomain to histone H3 methylated at Lys 27. Genes Dev 17, 1823-8 (2003).
Nielsen, P.R. et al. Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature 416, 103-7 (2002).
Flanagan, J.F. et al. Double chromodomains cooperate to recognize the methylated histone H3 tail. Nature 438, 1181-5 (2005).
Joshi, A.A. & Struhl, K. Eaf3 chromodomain interaction with methylated H3-K36 links histone deacetylation to Pol II elongation. Mol Cell 20, 971-8 (2005).
Bernstein, E. et al. Mouse polycomb proteins bind differentially to methylated histone H3 and RNA and are enriched in facultative heterochromatin. Mol Cell Biol 26, 2560-9 (2006).