Horton John R, Engstrom Amanda, Zoeller Elizabeth L, Liu Xu, Shanks John R, Zhang Xing, Johns Margaret A, Vertino Paula M, Fu Haian, Cheng Xiaodong
From the Departments of Biochemistry.
the Graduate Program in Biochemistry, Cell and Developmental Biology.
J Biol Chem. 2016 Feb 5;291(6):2631-46. doi: 10.1074/jbc.M115.698449. Epub 2015 Dec 8.
The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases remove methyl groups from tri- and dimethylated lysine 4 of histone H3. Accumulating evidence from primary tumors and model systems supports a role for KDM5A (JARID1A/RBP2) and KDM5B (JARID1B/PLU1) as oncogenic drivers. The KDM5 family is unique among the Jumonji domain-containing histone demethylases in that there is an atypical insertion of a DNA-binding ARID domain and a histone-binding PHD domain into the Jumonji domain, which separates the catalytic domain into two fragments (JmjN and JmjC). Here we demonstrate that internal deletion of the ARID and PHD1 domains has a negligible effect on in vitro enzymatic kinetics of the KDM5 family of enzymes. We present a crystal structure of the linked JmjN-JmjC domain from KDM5A, which reveals that the linked domain fully reconstitutes the cofactor (metal ion and α-ketoglutarate) binding characteristics of other structurally characterized Jumonji domain demethylases. Docking studies with GSK-J1, a selective inhibitor of the KDM6/KDM5 subfamilies, identify critical residues for binding of the inhibitor to the reconstituted KDM5 Jumonji domain. Further, we found that GSK-J1 inhibited the demethylase activity of KDM5C with 8.5-fold increased potency compared with that of KDM5B at 1 mm α-ketoglutarate. In contrast, JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) had the opposite effect and was ~8-fold more potent against KDM5B than against KDM5C. Interestingly, the relative selectivity of JIB-04 toward KDM5B over KDM5C in vitro translates to a ~10-50-fold greater growth-inhibitory activity against breast cancer cell lines. These data define the minimal requirements for enzymatic activity of the KDM5 family to be the linked JmjN-JmjC domain coupled with the immediate C-terminal helical zinc-binding domain and provide structural characterization of the linked JmjN-JmjC domain for the KDM5 family, which should prove useful in the design of KDM5 demethylase inhibitors with improved potency and selectivity.
KDM5/JARID1家族的Fe(II)和α-酮戊二酸依赖性去甲基化酶可去除组蛋白H3三甲基化和二甲基化赖氨酸4上的甲基基团。来自原发性肿瘤和模型系统的越来越多的证据支持KDM5A（JARID1A/RBP2）和KDM5B（JARID1B/PLU1）作为致癌驱动因子的作用。KDM5家族在含Jumonji结构域的组蛋白去甲基化酶中是独特的，因为在Jumonji结构域中有一个非典型的DNA结合ARID结构域和一个组蛋白结合PHD结构域插入，这将催化结构域分成两个片段（JmjN和JmjC）。在这里，我们证明ARID和PHD1结构域的内部缺失对KDM5家族酶的体外酶动力学影响可忽略不计。我们展示了KDM5A的连接JmjN-JmjC结构域的晶体结构，该结构表明连接结构域完全重构了其他结构特征明确的Jumonji结构域去甲基化酶的辅因子（金属离子和α-酮戊二酸）结合特性。用KDM6/KDM5亚家族的选择性抑制剂GSK-J1进行对接研究，确定了抑制剂与重构的KDM5 Jumonji结构域结合的关键残基。此外，我们发现，在1 mM α-酮戊二酸条件下，GSK-J1抑制KDM5C的去甲基化酶活性的效力比抑制KDM5B时高8.5倍。相比之下，JIB-04（Jumonji去甲基化酶超家族的泛抑制剂）则有相反的效果，对KDM5B的效力比对KDM5C高约8倍。有趣的是，JIB-04在体外对KDM5B相对于KDM5C的相对选择性转化为对乳腺癌细胞系的生长抑制活性高约10至50倍。这些数据确定了KDM5家族酶活性的最低要求是连接的JmjN-JmjC结构域与紧邻的C端螺旋锌结合结构域，并提供了KDM5家族连接的JmjN-JmjC结构域的结构特征，这在设计具有更高效力和选择性的KDM5去甲基化酶抑制剂方面应该会很有用。
