Human chromobox homolog 7

Target ID:

CBX7

Deposition Date:

Monday 25th February 2008

Families:

Methyl-lysine readers

Related Diseases:

CancerChromatin and epigenetics

Structure type:

apo

Download Coordinates:

2K1B

Authors:

Kaustov L, Lemak A, Quyang H, Gutmanas A, Fares C, Bountra C, Weigelt J, Loppnau P, Ravichandran M, Edwards AM, Min J, Arrowsmith CH

Site:

Toronto

ICM Datapack:

ICM Datapack

2K1B

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.
CBX7 is a Polycomb Group Chromodomain containing propteins, whose dysregulation has been linked with the aberrant proliferation of cancer cells. We solved the solution structure of the chromodomain of CBX7 by NMR. Its structure shows that CBX7 chromodomain has a high structure similarity to that of HP1 and Polycomb.

Materials and Methods

CBX7

PDB:2K1B

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_783640
Entry Clone Source:MGC AT75-D1 (BC051773)
SGC Clone Accession:
Tag:MHHHHHHSSGRENLYFQG
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfqgEQVFAVESIRKKRVRKGKVEYLVKWKGWPPKYSTWEPEEHILDPRLVMAYEEKEE
Vector:p28a-mhl

Growth

Medium:
Antibiotics:
Procedure:A 250 ml flask containing M9 base minimum media (with ¹³C-glucose and ¹⁵N (NH₄)₂SO₄) supplemented with 50 ug/ ml kanamycin (BioShop Canada KAN 201) was inoculated from a fresh transformed plate. 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 minimum media supplemented 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) = 2 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 4 degC. 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.

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 eluate 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 of 4-5 mg/mL.
Ligand
MassSpec:
Crystallization:
NMR Spectroscopy:All of the spectra were recorded at 25 °C on Varian INOVA 600 MHz and Bruker Avance 500 MHz spectrometers equipped with triple resonance 1H/13C/15N cold and cryoprobes, respectively. The final NMR samples contained 90% H2O, 10% D2O with a protein concentration ranging between 0.5 and 0.7 mM. The spectra were processed with NMRPipe software and analyzed with the SPARKY program (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco). Linear prediction in the 13C and 15N dimensions was used to improve the digital resolution. Distance restraints for structure calculations were obtained from 13C-edited NOESY-HSQC (m = 120 ms) and 15N-edited NOESY-HSQC (m = 150 ms) experiments. NOESY spectra were analyzed with the SPARKY program. Structure calculations were carried out using CNS version 1.1 with its standard annealing protocol. The 20 lowest energy structures were refined using CNS by performing a short constrained molecular dynamics simulation in explicit solvent.
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).