2. Academic Validation
  3. Structures of human ALKBH5 demethylase reveal a unique binding mode for specific single-stranded N6-methyladenosine RNA demethylation

Structures of human ALKBH5 demethylase reveal a unique binding mode for specific single-stranded N6-methyladenosine RNA demethylation

  • J Biol Chem. 2014 Jun 20;289(25):17299-311. doi: 10.1074/jbc.M114.550350.
Chao Xu 1,  Ke Liu 2,  Wolfram Tempel 1,  Marina Demetriades 3,  WeiShen Aik 3,  Christopher J Schofield 3,  Jinrong Min 4
Affiliations

Affiliations

  • 1 the Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
  • 2 the Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada, From the Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan 430079, China.
  • 3 the Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, United Kingdom, and.
  • 4 the Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada, From the Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, Wuhan 430079, China, the Department of Physiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada [email protected].
PMID: 24778178 DOI: 10.1074/jbc.M114.550350
Abstract

N(6)-Methyladenosine (m(6)A) is the most prevalent internal RNA modification in eukaryotes. ALKBH5 belongs to the AlkB family of dioxygenases and has been shown to specifically demethylate m(6)A in single-stranded RNA. Here we report crystal structures of ALKBH5 in the presence of either its cofactors or the ALKBH5 inhibitor citrate. Catalytic assays demonstrate that the ALKBH5 catalytic domain can demethylate both single-stranded RNA and single-stranded DNA. We identify the TCA cycle intermediate citrate as a modest inhibitor of ALKHB5 (IC50, ∼488 μm). The structural analysis reveals that a loop region of ALKBH5 is immobilized by a disulfide bond that apparently excludes the binding of dsDNA to ALKBH5. We identify the m(6)A binding pocket of ALKBH5 and the key residues involved in m(6)A recognition using mutagenesis and ITC binding experiments.

Keywords

Crystal Structure; Dioxygenase; Isothermal Titration Calorimetry (ITC); RNA Catalysis; RNA Methylation; Tricarboxylic Acid Cycle (TCA Cycle) (Krebs Cycle).

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