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.
Highlights
ALKBH5 catalyzes demethylation of m6A single-stranded RNA
By molecular modeling of the ALKBH5 substrate complex structure, we identify potential m6A binding residues, the identities of which are validated by mutagenesis and Isothermal titration calorimetry (ITC) binding experiments
The core of the ALKBH5 catalytic domain contains the double-stranded -helix (DSBH or jelly-roll) fold that is stereotypical of the 2OG oxygenases and consists of a total of 11  strands (1-11) and 5 ␣ helices (␣1-␣5) (Fig. 1)
Summary
ALKBH5 catalyzes demethylation of m6A single-stranded RNA (ssRNA). Results: ALKBH5 structures reveal the structural basis of its substrate selectivity and inhibition by citrate. The YTH domain containing proteins YTHDF1–3 were shown to recognize m6A containing singlestranded RNA [6, 11] Both the FTO and ALKBH5 RNA demethylases belong to the AlkB subfamily of the Fe(II)/2-oxoglutarate (2OG) dioxygenase superfamily. The Crystal Structures of ALKBH5 RNA Demethylase the human AlkB family members share the stereotypical 2OG oxygenase DSBH fold and have related 2OG and iron binding sites, their substrate selectivities differ. By molecular modeling of the ALKBH5 substrate complex structure, we identify potential m6A binding residues, the identities of which are validated by mutagenesis and ITC binding experiments During revision of this manuscript, two other manuscripts on human ALKBH5 structures were reported [38, 39]; their structural data and conclusions are consistent with our structural results, which are presented here
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