Jumonji domain of human Lysine-specific demethylase 8 (KDM8) in complex with N-oxalylglycine (NOG)

Target ID:

FLJ13798A

Deposition Date:

Monday 5th December 2011

Families:

Oxidoreductases

Structure type:

protein-ligand

Download Coordinates:

4AAP

Authors:

M.Vollmar,C.Johansson,T.Krojer,P.Canning,C.Allerston,A.Gadhave,F.von Delft,C.Bountra,C.H.Arrowsmith,J.Weigelt,A.Edwards,U.Oppermann

Site:

Oxford

4AAP

tabs

Structure Details

Post translational modifications of histone tails play a key role in chromatin architecture and dynamics (1). The methylation of lysine residues of histones H3 and H4 is critical to such biological processes as DNA repair and transcriptional activation, and the functional outcomes are context dependent and rely on the particular lysine residue and its methylation state (mono- di- or tri-methylated) (2). The recently discovered JMJD5, which also is called KDM8, belongs to the family of Fe(II)- and 2-oxoglutarate (2-OG) dependent histone demethylases that contains a jumonji-C domain (JmjC-domain). JMJD5 is shown to demethylate H3K36Me2 both in vivo and in vitro. The protein is over expressed in several cancer cells and suggested to be an important cell cycle regulator (3). Deletion of JMJD5 in mice causes severe growth retardation, resulting in embryonic lethality at the mid-gestation stage. This has suggested an important role of JMJD5 during embryonic cell proliferation by fine tuning the expression of Cdkn1a (4, 5). Recently, JMJD5 was also suggested to be an osteoclastogenic repressor by hydoxylating and thereby facilitating degradation of the NFATc1 protein (6). Thus, JMJD5 appears to have dual functions both as a histone demethylase and a protein hydroxylase.

Here we have determined the crystal structure of the catalytic a JmjC-domain in JMJD5 in complex with N-oxalylglycine (NOG), a 2-OG mimic, to 2.6 Å resolution.

Materials and Methods

FLJ13798 (4AAP) Materials & Methods

Entry Clone Source: MGC

Construct ID: FLJ13798A-c222

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Final protein sequence (Tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq^sMLE
KTVPRLHRPSLQHFREQFLVPGRPVI
LKGVADHWPCMQKWSLEYIQEIAGCR
TVPVEVGSRYTDEEWSQTLMTVNEFI
SKYIVNEPRDVGYLAQHQLFDQIPEL
KQDISIPDYCSLGDGEEEEITINAWF
GPQGTISPLHQDPQQNFLVQVMGRKY
IRLYSPQESGALYPHDTHLLHNTSQV
DVENPDLEKFPKFAKAPFLSCILSPG
EILFIPVKYWHYVRALDLSFSVSFWW
S

^ TEV protease recognition site

Tags and additions: Cleavable N-terminal His6 tag

Host: BL21(DE3)-R3-pRARE2

Growth Medium & Induction Protocol: Medium: TB supplemented with 50 µg/ml Kanamycin and 34 µg/ml chloramphenicol. 4 liter TB in two 4L flasks were inoculated with 40 ml overnight culture and grown at 37°C until OD₆₀₀ reached 2.5. The temperature was then decreased to 18°C and the protein expression induced with 0.1 mM IPTG over night. The cells were collected by centrifugation (4000 RPM, 30 minutes) and frozen at -80°C.

Extraction buffer, extraction method: The cell pellet was resuspended in a total volume of 200 ml lysis buffer and the cells disrupted by sonication. Nucleic acids and cell debris were removed by adding 0.15% PEI, followed by centrifugation for 60 minutes at 40 000xg. The supernatant was further clarified by filtration (0.20 µm).
Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 10 mM Imidazole, 5% glycerol, 3 U/ml of Benzonase, Complete® protease inhibitors (1 tablet/50 ml)

Column 1: Ni-sepharose, HisTrap FF, 5 ml (GE healthcare).

Column 1 Buffers:
Lysis buffer: 50 mM HEPES pH 8.0, 500 mM NaCl, 10 mM Imidazole, 5% glycerol.
Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM Imidazole, 5 % glycerol.
Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 300 mM Imidazole, 5 % glycerol.

Column 2: S200, (GE healthcare)

Column 2 Buffers:
Gel Filtration buffer: 10 mM HEPES pH 7.5, 500 mM NaCl, 5 % glycerol.

Column 2 Procedure: The cell extract was applied onto a 5 ml Ni-Sepharose FF gravity column equilibrated with lysis buffer. The column was subsequently washed with 10 volumes of lysis buffer followed by 10 wash buffer and protein eluted with 2x3 column volumes of elution buffer. All fractions were collected and analysed by 4-12% SDS-PAGE. The protein was concentrated in amicon 10 K to 5 ml, filtered through a 0.20 µm PVDF filter and applied onto a Superdex S200 column at 1.2 ml/min using an AKTA Xpress system. The eluted proteins were collected in 1.8 ml fractions and analysed by SDS-PAGE.

Column 3: Ni-Sepharose FF (TEV clean up)

Column 3 Buffers:
Wash buffer: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol

Column 3 Procedure: The N-terminal histidine-tag was cleaved with 150 µg of TEV protease per 10 mg protein at 4°C for 48 hours. The TEV cleaved protein was applied to a 0.5 ml Ni-sepharose column and the flow through collected. The column was washed with 5 ml buffer and the flow through and the wash fraction were analysed by SDS-PAGE.

Concentration: The protein was concentrated in Amicon 10K to 32 mg/ml. The protein concentration was determined spectrophotometrically using the predicted molar extinction coefficient 53400 (M^-1 cm^-1) and the predicted mass of 27735.7 Da

Mass spec characterization:
Expected: 27735.7
Observed: 27736.3

Crystallization: FLJ13798A was crystallised by vapor diffusion at 20°C from a sitting drop consisting of 100 nl protein (32 mg/ml) and 50 nl 16 % PEG3350, 7 mM NiCl₂, 7 mM MgCl₂, 0.1 M Hepes pH 7.3. The crystal was transferred to a cryo protectant composed of 33 % glycerol before flash-cooling in liquid nitrogen.

Data Collection:
Resolution: 2.60Å X-ray source: Diamond IO2

References

Bannister, A. J., and Kouzarides, T. (2011) Regulation of chromatin by histone modifications. Cell Res 21, 381-395
Kooistra, S. M., and Helin, K. (2012) Molecular mechanisms and potential functions of histone demethylases. Nat Rev Mol Cell Biol 13, 297-311
Hsia, D. A., Tepper, C. G., Pochampalli, M. R., Hsia, E. Y., Izumiya, C., Huerta, S. B., Wright, M. E., Chen, H. W., Kung, H. J., and Izumiya, Y. (2010) KDM8, a H3K36me2 histone demethylase that acts in the cyclin A1 coding region to regulate cancer cell proliferation. Proceedings of the National Academy of Sciences of the United States of America 107, 9671-9676
Ishimura, A., Minehata, K., Terashima, M., Kondoh, G., Hara, T., and Suzuki, T. (2012) Jmjd5, an H3K36me2 histone demethylase, modulates embryonic cell proliferation through the regulation of Cdkn1a expression. Development 139, 749-759
Oh, S., and Janknecht, R. (2012) Histone demethylase JMJD5 is essential for embryonic development. Biochemical and Biophysical Research Communications 420, 61-65
Youn, M. Y., Yokoyama, A., Fujiyama-Nakamura, S., Ohtake, F., Minehata, K., Yasuda, H., Suzuki, T., Kato, S., and Imai, Y. (2012) JMJD5, a Jumonji C (JmjC) Domain-containing Protein, Negatively Regulates Osteoclastogenesis by Facilitating NFATc1 Protein Degradation. The Journal of biological chemistry 287, 12994-13004