2. Academic Validation
  3. 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

  • J Biol Chem. 2014 Apr 25;289(17):11571-11583. doi: 10.1074/jbc.M113.546168.
Chong Feng 1,  Yang Liu 1,  Guoqiang Wang 1,  Zengqin Deng 1,  Qi Zhang 1,  Wei Wu 1,  Yufeng Tong 2,  Changmei Cheng 3,  Zhongzhou Chen 4
Affiliations

Affiliations

  • 1 State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.
  • 2 Structural Genomics Consortium, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada; Department of Pharmacology and Toxicology, University of Toronto, 101 College Street, Toronto, Ontario M5G 1L7, Canada.
  • 3 Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, China. Electronic address: chengcm@mail.tsinghua.edu.cn.
  • 4 State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China. Electronic address: chenzhongzhou@cau.edu.cn.
PMID: 24616105 DOI: 10.1074/jbc.M113.546168
Abstract

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 β-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 β-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 α-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.

Keywords

AlkB; Alkbh5; Crystal Structure; Drug Design; Enzyme Inhibitors; RNA Modification; Substrate Recognition; m6A Demethylase; mRNA.

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