Crystal structure of the RNA demethylase ALKBH5 from zebrafish

Abstract

ALKBH5, a member of AlkB family proteins, has been reported as a mammalian N(6)-methyladenosine (m(6)A) RNA demethylase. Here we report the crystal structure of zebrafish ALKBH5 (fALKBH5) with the resolution of 1.65Å. Structural superimposition shows that fALKBH5 is comprised of a conserved jelly-roll motif. However, it possesses a loop that interferes potential binding of a duplex nucleic acid substrate, suggesting an important role in substrate selection. In addition, several active site residues are different between the two known m(6)A RNA demethylases, ALKBH5 and FTO, which may result in their slightly different pathways of m(6)A demethylation.

Keywords: ALKBH5; Crystal structure; Demethylation; N(6)-Hydroxymethyladenosine.

Fig. 1

(A) MALDI-TOF/TOF mass spectrometry analysis. hALKBH5 and ΔfALKBH5 showed the same activity of m⁶A demethylation on ssRNA (ssRNA 5'-UAAGm⁶ACUCA-3') with loss of 14-Dalton of a methyl group after the reaction in both cases. By contrast, after treating FTO with ssRNA, two intermediates hm⁶A (m⁶A-2Da, lost a H₂O moiety during MALDI-TOF ionization) and fm⁶A (m⁶A+14Da) were observed. The two intermediates were not observed in the reaction with hALKBH5 and ΔfALKBH5 under the same condition. (B) HPLC chromatograms of digested nucleosides from m⁶A-containing ssRNA. HPLC confirmed the observation that after treatment with hALKBH5 and ΔfALKBH5, m⁶A underwent complete conversion to adenosine.

Fig. 2

The crystal structure of ΔfALKBH5. Two orthogonal views with catalytic core shown. The secondary structural elements are labeled α1-α3 for helices (colored cyan) and β1-β11 for strands (colored magenta).

Fig. 3

The sequence and structure alignment of AlkB family members (FTO (PDB ID: 3LFM), hALKBH2 (PDB ID: 3BTX), hALKBH3 (PDB ID: 2IUW), AlkB (PDB ID: 2FD8), hALKBH8 and fALKBH5). (A) Structure-based sequence alignment of AlkB family proteins. The five invariant residues are highlighted in red. The loop L1 in FTO, L2 in human and fish ALKBH5 as well as the lid in FTO, hALKBH2, hALKBH3 and AlkB are boxed off in red. The secondary structure of fALKBH5 is shown on top of the alignment. (B) Structural comparison of fALKBH5 and hALKBH2-DNA. The L2 loop (colored in blue) in fALKBH5 protrudes into the dsDNA strand (colored in orange) from the superimposed hALKBH2-dsDNA structure. (C) Structure alignment of fALKBH5 and FTO with the extra loop highlighted. (L1 in FTO is colored red, and L2 in fALKBH5 is colored blue.) (D) A hairpin creates a lid over the active site in FTO, AlkB, hALKBH2 and hALKBH3. The structure of fALKBH5 lacks such a lid. The lid is highlighted in a red box.

Fig. 4

The interaction network around Mn(II) and α-KG (A) or succinate (B). The coordinate bonds between Mn(II) and its ligands are shown with red dash lines, whereas the interactions between Arg245/Arg251 and α-KG/SIN are indicated in green. The distance between Arg251 and α-KG is 4.4 Å, indicating a weak interaction between them.

Fig. 5

Superimposition of residues near the catalytic core in fALKBH5 and FTO. The mononucleotide m³T in FTO is colored green and residues in fALKBH5 and FTO are labeled in cyan and red, respectively.