Structural and Biochemical Insights into Methylation Site and State Specificity of JMJD2 Lysine Demethylases.

[摘要] The human JMJD2/KDM4 family of histone lysine demethylases comprises four homologs: JMJD2A, JMJD2B, JMJD2C and JMJD2D. These enzymes have been implicated in a number of biological processes such as transcriptional activation, development and cell cycle control. The biological functions of these enzymes are defined by their distinct methylation site and state specificities. JMJD2A, JMJD2B and JMJD2C display dual specificity for trimethylated histone H3 Lys9 and Lys36 (H3K9me3 and H3K36me3), whereas JMJD2D is specific for H3K9me3. Furthermore, while most JMJD2 homologs are predominantly trimethyllysine-specific, JMJD2D can demethylate both tri- and dimethyllysines. To enable quantitative kinetic studies of JMJD2 demethylases, we developed and applied a new affinity purification protocol that minimizes contamination by transition state metals. In order to delineate the molecular basis of site and state specific demethylation by the JMJD2 enzymes, we determined the first crystal structure of JMJD2D in the apoenzyme form and in a ternary complex with 2-OG and an H3K9me3 peptide. Our site specificity studies with JMJD2A and JMJD2D revealed surprising differences in H3K9me3 recognition by these enzymes despite the overall similarity in the substrate binding conformation. In addition, our docking studies with H3K36me3 and biochemical analysis with histone H3 hybrid peptides underscored the role of steric clashes, electrostatic clashes and loss of productive hydrogen bonds in occluding recognition of the H3K36me3 site by JMJD2D.Our structural and biochemical analysis of the active site also revealed the basis for differential state specificity in the JMJD2 enzymes and highlighted the role of CH---O hydrogen bonds in di- and trimethyllysine substrate recognition. Together, these structural and biochemical studies elucidate the molecular basis of the different substrate specificities within the JMJD2 family, which is not only key to understanding their distinct biological functions but will also aid in the structure-based design of selective inhibitors of JMJD2 enzymes implicated in disease.

[发布机构] University of Michigan

[效力级别] Biological Chemistry

[关键词] Substrate Specificity of JMJD2 Lysine Demethylases;Biological Chemistry;Chemistry;Health Sciences;Science;Biological Chemistry