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

Chao Xu, Ke Liu, Wolfram Tempel, Marina Demetriades, Wei Shen Aik, Christopher J. Schofield, Jinrong Min*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

141 Citations (Scopus)

Abstract

N6-Methyladenosine (m6A) is the most prevalent internal RNA modification in eukaryotes. ALKBH5 belongs to the AlkB family of dioxygenases and has been shown to specifically demethylate m6A 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 m6A binding pocket of ALKBH5 and the key residues involved in m6A recognition using mutagenesis and ITC binding experiments.

Original languageEnglish
Pages (from-to)17299-17311
Number of pages13
JournalJournal of Biological Chemistry
Volume289
Issue number25
DOIs
Publication statusPublished - 20 Jun 2014

Scopus Subject Areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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