Crystal Structure and Functional Analysis of JMJD5 Indicate an Alternate Specificity and Function

Abstract

JMJD5 is a Jumonji C (JmjC) protein that has been implicated in breast cancer tumorigenesis, circadian rhythm regulation, embryological development, and osteoclastogenesis. Recently, JMJD5 (also called KDM8) has been reported to demethylate dimethylated Lys-36 in histone H3 (H3K36me2), regulating genes that control cell cycle progression. Here, we report high-resolution crystal structures of the human JMJD5 catalytic domain in complex with the substrate 2-oxoglutarate (2-OG) and the inhibitor N-oxalylglycine (NOG). The structures reveal a β-barrel fold that is conserved in the JmjC family and a long shallow cleft that opens into the enzyme's active site. A comparison with other JmjC enzymes illustrates that JMJD5 shares sequence and structural homology with the asparaginyl and histidinyl hydroxylase FIH-1 (factor inhibiting hypoxia-inducible factor 1 [HIF-1]), the lysyl hydroxylase JMJD6, and the RNA hydroxylase TYW5 but displays limited homology to JmjC lysine demethylases (KDMs). Contrary to previous findings, biochemical assays indicate that JMJD5 does not display demethylase activity toward methylated H3K36 nor toward the other methyllysines in the N-terminal tails of histones H3 and H4. Together, these results imply that JMJD5 participates in roles independent of histone demethylation and may function as a protein hydroxylase given its structural homology with FIH-1 and JMJD6.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00513-12.

ACKNOWLEDGMENTS

We thank Brittany Bowman for assistance in protein purification and crystallization and Henriette Remmer (University of Michigan Medical School, Protein Structure Core) and Ted Huston (University of Michigan, Department of Geological Sciences, W. M. Keck Elemental Geochemistry Laboratory) for mass spectrometry assistance. We acknowledge Daniel Bochar for providing useful comments on the manuscript. We also thank Chris Lima for providing the Smt3-Ulp1 expression system.

Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under contract DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor for the support of this research program (grant 085P1000817). This work utilized the Protein Structure Core of the Michigan Diabetes Research and Training Center funded by grant DK020572 from the National Institute of Diabetes and Digestive and Kidney Disease. S.K. received support through research grants from the University of Michigan's Rackham Graduate School and Department of Biological Chemistry. This work was supported by funding to R.C.T. from the University of Michigan's Biomedical Research Council and the Office of the Vice President Research.