Human jumonji domain containing 2E in complex with 2,4-PDCA

Target ID:

LOC390245C

Aliases:

JMJD2E

Deposition Date:

Friday 31st October 2008

Families:

Miscellaneous

Related Diseases:

Chromatin and epigenetics

Structure type:

protein-ligand

Download Coordinates:

2W2I

Authors:

W.W.Yue, S.S.Ng, E.Ugochukwu, M.A.McDonough, A.C.W.Pike, P.Filippakopoulos, F.von Delft, C.Arrowsmith, J.Weigelt, A.Edwards, C.Bountra, C.J.Schofield, U.Oppermann

Site:

Oxford

ICM Datapack:

ICM Datapack

2W2I

tabs

Structure Details

Covalent modification of histone tails plays a fundamental role in regulation of functional chromatin organization and constitutes the "histone code" involved in epigenetic regulation. Methylation of lysyl residues of histones H3 and H4 is critical to many biological processes such as heterochromatin formation, X chromosome inactivation, genome imprinting, DNA repair and transcriptional regulation. The effect of histone lysyl modification is context dependent and relies on the particular residue and the methylation state. In most instances lysyl methylation at H3K9, H3K27 and H4K20 is associated with transcriptionally silent regions, whereas methylation of H3K4, H3K36 and H3K79 appears in transcriptionally active chromatin. Methylated lysyl residues in histones recruit adaptor molecules that dictate the specific effects of methylation, e.g. methylated H3K9 associates with HP1 allowing heterochromatin formation and silencing. Furthermore, the methylation state (mono, di or tri) of Lys confers specificity to recruitment of chromatin remodeling components.

Until 2006, histone lysine methylation was considered as a stable epigenetic marker, however the discovery of enzymes capable of demethylating methylated lysyl groups dramatically changed this view. Recently two processes have been identified that catalyse demethylation of Nε-methylated lysyl residues. The FAD dependent amine oxidase LSD 1 (Lysyl demethylase 1) catalyses demethylation of mono and di-methylated, but not trimethylated lysyl residues via an imine intermediate. The recent discovery that members of the Fe(II) and 2-oxoglutarate (2-OG) dependent oxygenases demethylate histone lysyl residues opened a new perspective on epigenetic regulation. To this end, catalytic activity towards methylated histone lysyl residues has been shown for members of three distinct subfamilies: JMJD1A of the JHDM2 subfamily demethylates mono and dimethylated H3K9, FBXL10 and FBXL11 of the JHDM1 subfamily catalyze demethylation of H3K36, JMJD3 catalyses demethylation of H3K27 and members of the JHDM3/JMJD2 subfamily display specificity towards tri and dimethylated states of H3K9 and H3K36.

JMJD2E(KDM5E) has been identified as a pseudogene, it displays similar specificity to that of JMJD2D(KDM5D) a H3K9 demethylase. In comparison to other JMJD2 family members JMJD2E is capable of very rapidly demethylating H3 lysine 9. This makes JMJD2E(KDM5E) an ideal model protein for elucidating the specificity of demethylation.JMJD2E(KDM5E) containing nickel in place of iron (II) has been co-crystallised with 2,4 pyridinedicarboxylic acid (2,4-PDCA). 2,4-PDCA complexes in the co-factor part of the active site and adopts the same coordination as that found in JMJD2A(KDM5A) in complex with 2,4-PDCA.

Materials and Methods

Entry Clone Source: Synthetic

Entry Clone Accession: n/a

SGC Construct ID: LOC390245C-c103

GenBank GI number: gi|89034221

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

Tag and Additions: N-Terminal TEV cleavable 6His tag - mhhhhhhssgvdltgtenlyfq*s(m), TEV cleaves at *.

Protein Sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq*sMKSVHS
SPQNTSHTIMTFYPTMEEFADFNTYVAYM
ESQGAHQAGLAKVIPPKEWKARQMYDDIE
DILIATPLQQVTSGQGGVFTQYHKKKKAM
RVGQYRRLANSKKYQTPPHQNFADLEQRY
WKSHPGNPPIYGADISGSLFEESTKQWNL
GHLGTILDLLEQECGVVIEGVNTPYLYFG
MWKTTFAWHTEDMDLYSINYLHFGEPKTW
YVVPPEHGQHLERLARELFPDISRGCEAF
LRHKVALISPTVLKENGIPFNCMTQEAGE
FMVTFPYGYHAGFNHGFNCAEAINFATPR
WIDYGKMASQCSCGESTVTFSMDPFVRIV
QPESYELWKHR

Host: E. coli BL21(DE3)-R3 containing pRARE2 plasmid

Growth Medium, induction protocol: Medium: Terrific Broth +50 µg/ml Kanamycin, +34 µg/ml chloramphenicol. 6 x 1 litres TB in 2.5 L baffled flasks, each flask was inoculated with 5 ml overnight culture and grown at 37°C. The protein expression was induced with 0.2 mM IPTG at OD₆₀₀= 0.8 for 18 hours at 18°C. The cells were harvested by centrifugation, resuspended inlysis buffer (see below) and frozen at -80°C.

Lysis buffer, lysis method: Lysis Buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM Imidazole, 5% Glycerol, 0.5 mM TCEP, Complete Protease Inhibitor (Roche Biochemicals). Frozen cell pellets were thawed and lysed by high pressure homogenisation (15kpsi), viscosity was reduced by 3 further passes through the homogeniser. Cell debris were removed by centrifugation at 40,000 x g for 60 minutes. The lysate was then diluted 1:1 with Lysis buffer

All chromatography performed at 4°C.

Column 1: 5 ml bed volume Ni Sepharose 6 FF gravity column (GE/Amersham Biosciences)

Procedure: The gravity column was pre-equilibrated with IMAC Binding Buffer (Lysis Buffer without Complete PI), the lysate was applied to the column and allowed to flow through completely. The column was then washed with 2 x 10 ml of IMAC BInding Buffer. The column was then washed with 4 x 50 ml of IMAC Wash Buffer (IMAC Binding Buffer with 40 mM Imidazole). The column was then eluted with IMAC Elution Buffer (IMAC Binding Buffer with 250 mM Imidazole).

TEV digest: TEV protease was added to the eluated protein and incubated overnight at 4°C.

Column 2: HiPrep 26/1 Desalting

Procedure: The protein was then desalted into IMAC Binding Buffer using a HiPrep 26/10 desalting column on an AKTAPrime (GE/Amersham Biosciences).

Column 3: 2 ml bed volume Ni Sepharose 6 FF gravity column (GE/Amersham Biosciences)

Procedure: The protein was then applied to the column and the flow through collected. The column was washed with 2 x 5 ml of IMAC Binding Buffer and the flow through collected. The flow through was concentrated to 5 ml.

Column 4: HiLoad 16/60 SuperDex 200 Prep Grade

Procedure: The gel filtration column was equilibrated with 10 mM HEPES pH7.5, 500 mM NaCl, 5% Glycerol. The protein was applied to column and fractions collected. Fractions were analysed by SDS-PAGE, fractions were pooled based on SDS-PAGE purity. TCEP-HCl was added to 0.5 mM, slight precipitation was seen, the protein was centrifuged to remove precipitate and was immediately desalted.

Column 5: HiPrep 26/1 Desalting

Procedure: The desalting column was equilibrated with 10 mM HEPES pH7.5, 500 mM NaCl, 5% Glycerol. No TCEP-HCl added.

Concentration: The protein was concentrated to 10 mg/ml by A280 with a Millipore/Amicon Ultracel 30 kD MWCO ultrafiltration concentrator.

Mass spectrometry characterisation: The mass determined for the protein was 38681 Da, in agreement for the predicted mass for the cleaved protein.

Crystallisation: Crystals were grown by vapour diffusion at 4°C. 25 µl of protein had 2.5 µl of 25 mM 2,4 pyridine dicarboxylic acid added in 50 mM HEPES pH 7.5 added. A sitting drop consisting of 134 nl of protein + 66 nl of well solution was equilibrated against 0.1 M citrate pH 5.5, 20% PEG3350, 2 mM NiCl2. Cryo-protection of the crystals used 0.1 M citrate pH 5.5, 20% PEG3350, 2 mM NiCl2, 25% glycerol and 2.5 mM 2,4-PDCA.

Data Collection: Resolution: 2.1 Å; X-ray source: Synchrotron SLS-X10SA