Crystal Structures of the Human RNA Demethylase Alkbh5 Reveal Basis for Substrate Recognition
Crystal Structures of the Human RNA Demethylase Alkbh5 Reveal Basis for Substrate Recognition
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人类 RNA 去甲基化酶 Alkbh5 的晶体结构揭示了底物识别的基础
DOI：
10.1074/jbc.m113.546168
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发表时间：
2014-04-25
影响因子：
4.8
通讯作者：
Chen, Zhongzhou
中科院分区：
文献类型：
--
作者：
Feng, Chong;Liu, Yang;Chen, Zhongzhou
背景：人类AlkB同源物5 （Alkbh5）是一种RNA去甲基化酶，可以消除m(6)A修饰。结果：确定了酶活性Alkbh5与辅助因子或小分子复合物的晶体结构。结论：结构和活性分析表明，Alkbh5强烈倾向于单链寡核苷酸和小分子抑制剂。意义：Alkbh5的结构揭示了基于结构设计选择性抑制剂的潜力。腺苷的n- 6甲基化是真核生物mRNA和长链非编码RNA中最普遍和最丰富的核苷修饰。这种修饰在mRNA翻译和RNA代谢的调控中起着重要作用。最近，人类AlkB同源物5 （Alkbh5）和脂肪质量和肥胖相关蛋白（FTO）被证明可以消除mRNA上的甲基修饰。在这里，我们报道了Alkbh5在不同配体的配合物中催化核心的五种高分辨率晶体结构。与其他AlkB蛋白相比，Alkbh5在该蛋白家族典型的保守双链螺旋折叠上显示出几个独特的结构特征。在这些独特的特征中，Alkbh5的一个独特的盖子区域在底物识别和催化中起着至关重要的作用。Cys-230和Cys-267之间意想不到的二硫键对于Alkbh5与单链RNA/DNA的选择性结合至关重要，通过将翻转基序带入中心螺旋折叠。我们基于几个结构导向的位点导向突变体的去甲基化活性测定生成了Alkbh5的底物结合模型。利用各种-酮戊二酸类似物进行的晶体学和生化研究表明，Alkbh5的活性位点腔比FTO的小得多，并且优先结合小分子抑制剂。综上所述，我们的发现为理解Alkbh5的底物识别特异性提供了结构基础，并为针对AlkB成员的选择性药物设计提供了基础。
Background: Human AlkB homolog 5 (Alkbh5) is an RNA demethylase that erases m(6)A modification. Results: Crystal structures of an enzymatically active Alkbh5 construct in complex with cofactors or small molecules were determined. Conclusion: Structure and activity analyses showed that Alkbh5 strongly prefers single-stranded oligos and small molecule inhibitors. Significance: The Alkbh5 structure reveals potential for structure-based design of selective inhibitors. N-6-Methylation of adenosine is the most ubiquitous and abundant modification of nucleoside in eukaryotic mRNA and long non-coding RNA. This modification plays an essential role in the regulation of mRNA translation and RNA metabolism. Recently, human AlkB homolog 5 (Alkbh5) and fat mass- and obesity-associated protein (FTO) were shown to erase this methyl modification on mRNA. Here, we report five high resolution crystal structures of the catalytic core of Alkbh5 in complex with different ligands. Compared with other AlkB proteins, Alkbh5 displays several unique structural features on top of the conserved double-stranded -helix fold typical of this protein family. Among the unique features, a distinct lid region of Alkbh5 plays a vital role in substrate recognition and catalysis. An unexpected disulfide bond between Cys-230 and Cys-267 is crucial for the selective binding of Alkbh5 to single-stranded RNA/DNA by bringing a flipping motif toward the central -helix fold. We generated a substrate binding model of Alkbh5 based on a demethylation activity assay of several structure-guided site-directed mutants. Crystallographic and biochemical studies using various analogs of -ketoglutarate revealed that the active site cavity of Alkbh5 is much smaller than that of FTO and preferentially binds small molecule inhibitors. Taken together, our findings provide a structural basis for understanding the substrate recognition specificity of Alkbh5 and offer a foundation for selective drug design against AlkB members.