Human jumonji domain containing 2A in complex with dimethylated H3K9 peptide

Target ID:

JMJD2AA

Aliases:

JHDM3AJMJD2JMJD2AKDM4AKIAA0677

Deposition Date:

Monday 19th February 2007

Families:

Oxidoreductases

Related Diseases:

Chromatin and epigeneticsCancer

Structure type:

apo

Download Coordinates:

2OX0

Authors:

E.S.PILKA,S.S.NG,K.L.KAVANAGH,M.A.MCDONOUGH,P.SAVITSKY,F.VON DELFT,C.H.ARROWSMITH,J.WEIGELT,A.EDWARDS,M.SUNDSTROM,C.J.SCHOFIELD,U.OPPERMANN,STRUCTURAL GENOMICS CONSORTIUM(SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2OX0

tabs

Structure Details

Covalent modification of histone tails plays a fundamental role in regulation of functional chromatin organization and constitutes the "histone code" critical 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 (2OG) 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, and members of the JHDM3/JMJD2 subfamily display specificity towards tri and dimethylated states of H3K9 and H3K36.

We have now determined several dead end complex structures of human JMJD2A with Ni(II) (replacing the Fe(II)), N-oxalylglycine (replacing 2OG) and different H3K9 and H3K36 substrate peptides in different methylation states.

Materials and Methods

Enrty Clone Source: MGC

Entry Clone Accession: BC002558

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

Tags and Additions: N-terminal TEV cleavable 6His tag - mhhhhhhssgvdlgtenlyfq*s(m), cleaves at *.

Protein Sequence:
mhhhhhhssgvdlgtenlyfqsMASESET
LNPSARIMTFYPTMEEFRNFSRYIAYIES
QGAHRAGLAKVVPPKEWKPRASYDDIDDL
VIPAPIQQLVTGQSGLFTQYNIQKKAMTV
REFRKIANSDKYCTPRYSEFEELERKYWK
NLTFNPPIYGADVNGTLYEKHVDEWNIGR
LRTILDLVEKESGITIEGVNTPYLYFGMW
KTSFAWHTEDMDLYSINYLHFGEPKSWYS
VPPEHGKRLERLAKGFFPGSAQSCEAFLR
HKMTLISPLMLKKYGIPFDKVTQEAGEFM
ITFPYGYHAGFNHGFNCAESTNFATRRWI
EYGKQAVLCSCRKDMVKISMDVFVRKFQP
ERYKLWKAGKDNTVIDHTLPTPEAAEFLK
ESEL

Host : E. coli BL21(DE3)-R3

Growth medium, induction protocol : Medium: TB + 50 µg/ml Kanamycin + 34 µg/ml chloramp. 12 x 1 liter TB in 2.5-L baffled flasks were 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 h at 18°C. The cells were collected by centrifugation and frozen at -80°C.

Extraction buffer, extraction method : Lysis buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP and PMSF 0.5 mM. Frozen cell pellets were thawed and resuspended in a total volume of 400 ml lysis buffer. The cells were disrupted by high pressure homogenisation (15 kpsi) followed by sonication. Cell debris were removed by centrifugation for 60 minutes at 40 000 x g.

Column 1 : Ni-affinity, HisTrap FF Crude, 1 ml (GE/Amersham Biosciences)

Procedure: The cell extract was loaded on the column at 0.8 ml/minute on an AKTA-express system (GE/Amersham). The column was then washed with 10 volumes of lysis buffer, 50 volumes of wash buffer (lysis buffer with 40 mM imidazole), and then eluted with elution buffer (lysis buffer with 250 mM imidazole) at 0.8 ml/min. The eluted peak of A280 was automatically collected.

Column 2 : Gel Filtration, Hiload 16/60 Superdex 200 prep grade, 120 ml (GE/ Amersham Biosciences)

Buffers: 10 mM HEPES, pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP

Procedure: The eluted fractions from the Ni-affinity Histrap column were loaded on the gel filtration column at 1.0 ml/min. Eluted proteins were collected in 1.8 ml fractions. The target protein pool had EDTA added to a final concentration of 1 mM and was left overnight.

Column 3 : HiPrep Desalting 26/10, Sephadex G-25 Fine, 57 ml (GE/ Amersham Biosciences)

Procedure: The protein pool from the gel filtration step was applied to the column which had been equilibrated into 50 mM tris-HCl pH 8.5, 50 mM NaCl. The resulting protein pool was 0.2 µm filtered through a cellulose acetate filter.

Column 4 : 5 ml HiTrap Q Sepharose High Performance (GE/ Amersham Biosciences)

Procedure: The protein pool was applied to the ion exchange column and washed with column volumes of 50 mM tris-HCl pH 8.5, 50 mM NaCl. The column was eluted with a 20 column volume gradient from 50-500 mM NaCl in 50 mM tris-HCl pH 8.5, collecting 1.8 ml fractions. The fractions were analysed by SDS-PAGE and the protein pooled based on fraction purity.

Column 5: HiPrep Desalting 26/10, Sephadex G-25 Fine, 57 ml (GE/ Amersham Biosciences)

Procedure: The protein pool from the ion exchnage step was applied to the column which had been equilibrated into 10 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP.

Concentration: The protein was concentrated using an Amicon Ultracel centrifugal concentrator (10 kDa MWCO) to 11 mg/ml by A280.

Mass spec characterisation: The mass determined for JMJD2AA-p020 was 44266 Da, in agreement with the predicted mass for the his-tagged protein.

Crystallisation: Crystals were grown by vapor diffusion at 4°C. A sitting drop consisting of 100 nl protein (11 mg/ml) + 10 mM N-oxalylglycine, 5 mM peptide and 50 nl well solution was equilibrated against well solution containing 0.1 M citrate pH 5.5, 20% PEG 3350, 4 mM NiCl 2 . Cryo-protection of the crystals used 0.1 M citrate pH 5.5, 20% PEG 3350, 4 mM NiCl 2 , 25% glycerol, 10 mM N-oxalylglycine and 30 mM peptide.

Data Collection: Resolution: 2.3 Å; X-ray source: Rotating anode, Rigaku FR-E superbright.