title: Crystal structures of the human RNA demethylase Alkbh5 reveal basis for substrate recognition.
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.
authors.affiliation: State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.
authors.forename: Chong
authors.initials: C
authors.lastname: Feng
authors.affiliation: State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.
authors.forename: Zengqin
authors.initials: Z
authors.lastname: Deng
authors.affiliation: State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.affiliation: 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.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Tong
authors.affiliation: 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.
authors.forename: Changmei
authors.initials: C
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China. Electronic address: chenzhongzhou@cau.edu.cn.
authors.forename: Zhongzhou
authors.initials: Z
authors.lastname: Chen
mesh_terms: D000071502:AlkB Homolog 5, RNA Demethylase; D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D049308:Dioxygenases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D008565:Membrane Proteins; D049308:Dioxygenases; C559920:ALKBH5 protein, human; D000071502:AlkB Homolog 5, RNA Demethylase
keywords: AlkB; Alkbh5; Crystal Structure; Drug Design; Enzyme Inhibitors; RNA Modification; Substrate Recognition; m6A Demethylase; mRNA