JMJD2A: Human jumonji domain containing 2A

PDB Code: 2OQ6 , 2OQ7 , 2OS2 , 2OT7 , 2OX0 , 2VD7

Ng, S.S., Pilka, E.S., Kavanagh, K.L., McDonough, M.A., Savitsky, P., von Delft, F., Gileadi, O., Lee, W.H., Muller, S., Marsden, B.D., Schofield, C.J., Oppermann, U.

Datapack version: 2 (built on 14.Jul.08)

Description

Covalent modification of histone tails plays a fundamental role in regulation of functional chromatin organization and constitutes the "histone code" critical in epigenetic regulation[1]. Methylation of lysyl residues of histones H3 and H4 is fundamental 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[2]. 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[3]. Furthermore, the methylation state (mono, di or tri) of Lys confers specificity to recruitment of chromatin remodeling components.

Until recently, 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. To date 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[4, 5]. 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 four distinct subfamilies: JMJD1A of the JHDM2 subfamily demethylates mono and dimethylated H3K9[6], FBXL10 and FBXL11 of the JHDM1 subfamily catalyze demethylation of H3K36[7], JARID members demethylate trimethylated H3K4, UTX and UTY demethylate H3K27 residues, and members of the JHDM3/JMJD2 subfamily such as JMJD2A display specificity towards tri and dimethylated states of H3K9 and H3K36[8, 9].

At present, no diseases have been directly linked to JMJD2A. However, its association with retinoblastoma binding protein (pRb) and histone deacetylases (HDACs), and involvement in pRb-mediated repression of E2F-regulated promoters imply an important role for this protein in cell proliferation and oncogenesis[10]. The closely related paralog JMJD2C (GASC1) has been shown to be upregulated in oesophageal squamous carcinomas[11] and has been suggested as a possible target in cancer treatment.

We have now determined several inhibitory dead-end complex structures of human JMJD2A with Ni(II)(replacing the endogenous Fe(II)), N-oxalylglycine (replacing the cosubstrate 2OG) with different H3K9 and H3K36 substrate peptides in different methylation states[12].

Structural features

General architecture : As seen in other 2-oxoglutarate dependent oxygenases, the core of the protein consists of a double stranded β-helix (DSBH) fold, also known as a “beta jelly roll”. In JMJD2A the DSBH is present in the JmjC domain and together with the JmjN domain at the N-terminus, forms a catalytically active domain, capable of demethylating modified histone peptides.

(TIP 1: the background colour can be changed to [WHITE] or [BLACK] )

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Substrate specificity of JMJD2A: A zinc binding site appears to be involved in peptide recognition and consists of residues His240, Cys234 from the JmjC domain and Cys308 and Cys306 from the C-terminal domain. The backbone carbonyl of His240 forms a hydrogen bond with backbone amide of Gly13 of H3K9 peptide, and interactions by Lys241 and Arg309 which flank the Zn binding site also point to the substrate recognition site.
The H3K9 substrate binds approximately in a broad “W”-shaped conformation in which residues downstream of Lys9 interact with the substrate pocket. The bound H3K36 adopts a tighter bend than in the H3K9 substrate leading to a “U”-shaped binding conformation. With the H3K9 substrate the H3Ser10 side-chain and main-chain H3Gly12 amide are positioned to form an intra-substrate hydrogen bond that will stabilize the bent conformation, shown by a Ser10Ala H3K9(me3) mutant peptide that no longer gets demethylated [12]. In addition, phosphorylation at Ser10 prohibits demethylase activity [12], highlighting the fact that phosphorylation and methylation states represent a “switch” mechanism between gene silencing and activation [11]. In summary, peptide substrate recognition of JMJD2A is achieved through few specific side-chain interactions, which is unusual for a sequence-specific enzyme.

Active site : The active site is located at one end of the beta sheets. Residues that coordinate the nickel include His188 and Glu190, which are part of a conserved HXD/E motif, as well as His276. A water molecule and two N-oxalylglycine (N-OG) oxygens complete the octahedral coordination. The N-OG forms hydrogen bonds with Tyr132, Asn198 and Lys206 side chains.
In the substrate peptide containing structures, the methylated lysine side-chains are superimposable. The histone peptides in the structures bend around Asp311 which is present on a flexible loop between helices α9 and α10 and this conformation of the substrate allows the side-chain lysine to enter the active-site.

In both H3K9 and H3K36 tri-methyl peptide structures the substrate pocket consists of the following amino acid side chains which interact with the methylated lysine: Tyr177, Glu190, Ser288 and Asn290 and Gly170. The phenyl ring of Tyr175 aligns the substrate towards the active-site, and the Glu190 carboxylate oxygen not involved in the metal coordination is pointed towards the tri-methyl group. The three methyl groups are arranged in a manner, that one is oriented towards Asn290, the second towards the Ni(II), (which substitutes for Fe(II)), and the third towards Tyr177. This arrangement enables hydroxylation of the methyl group by a highly reactive oxoferryl species leading to a hemiaminal that rearranges under the loss of formaldehyde to form the demthylated lysyl product [12].

We also solved H3K9 peptide structures in the di- and mono-methyl states; in each the overall peptide binding is similar to the H3K9 trimethylated form. Here the single methyl group of the monomethyl lysine occupies a position that is directed away from the metal. In this structure, two additional waters are observed close to the positions occupied by the other methyl groups in the trimethyl structures. One water is within hydrogen-bonding distance of the Gly170 and Ser288 main-chain carbonyls, while the water in the other site could interact with the hydroxyls of Tyr177 and Ser288 and the water involved in metal coordination [12].

In the case of the H3K9(Me2) structure , the dimethyl lysine side-chain is observed primarily in a catalytically non-productive conformation. The electron density for the peptide bound indicates two possible conformations ; one adopts a catalytically productive orientation with a methyl groups occupying the productive site as in the trimethyl structure. In each conformation a single water molecule, similarly positioned to one of the water positions in the monomethyl structure, occupies the position of the absent methyl group. This observation may explain the considerably lower activity observed towards the dimethylated peptide [12].

JMJD2A 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. 2,4-PDCA adopts a distinct orientation compared to 2-OG and N-oxalylglycine. 2,4-PDCA has been demonstrated to inhibit 2-OG dependant oxygenases such as prolyl-4-hydroxylase [13,14].

Note: The target annotations and structure descriptions within this datapack are compiled by our Principal Investigators and are not peer-reviewed. If you find anything in the annotations that is not accurate, please notify us using the our on-line feedback page or send an e-mail to isee@sgc.ox.ac.uk .

References

1. Strahl, B.D. and C.D. Allis, The language of covalent histone modifications. Nature, 2000. 403 (6765): p. 41-45.
2. Martin, C. and Y. Zhang, The diverse functions of histone lysine methylation. Nat Rev Mol Cell Biol, 2005. 6 (11): p. 838-49.
3. Snowden, A.W., et al., Gene-specific targeting of H3K9 methylation is sufficient for initiating repression in vivo. Curr Biol, 2002. 12 (24): p. 2159-66.
4. Wang, Y., et al., Human PAD4 regulates histone arginine methylation levels via demethylimination. Science, 2004. 306 (5694): p. 279-83.
5. Shi, Y., et al., Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell, 2004. 119 (7): p. 941-53.
6. Yamane, K., et al., JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor. Cell, 2006. 125 (3): p. 483-95.
7. Tsukada, Y., et al., Histone demethylation by a family of JmjC domain-containing proteins. Nature, 2006. 439 (7078): p. 811-6.
8. Whetstine, J.R., et al., Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell, 2006. 125 (3): p. 467-81.
9. Klose, R.J., et al., The transcriptional repressor JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine 36. Nature, 2006. 442 (7100): p. 312-6.
10. Gray, S.G., et al., Functional Characterization of JMJD2A, a Histone Deacetylase- and Retinoblastoma-binding Protein. J. Biol. Chem., 2005. 280 (31): p. 28507-28518.
11. Cloos, P.A., et al., The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3. Nature, 2006. 442 (7100): p. 307-11.
12. Ng, S.S. and e. al., Crystal structures of histone demethylase JMJD2A reveal basis for substrate specificity. Nature, 2007. 448: 87-91. 13. Majamaa K. et al. The 2-oxoglutarate binding site of prolyl 4-hydroxylase. Identification of distinct subsites and evidence for 2-oxoglutarate decarboxylation in a ligand reaction at the enzyme-bound ferrous ion. Eur J Biochem. 1984 Jan 16;138(2):239-45.
14. Majamaa K. et al. Differences between collagen hydroxylases and 2-oxoglutarate dehydrogenase in their inhibition by structural analogues of 2-oxoglutarate. Biochem J. 1985 Jul 1;229(1):127-33.