7WL0

Crystal structure of human ALKBH5 in complex with N-oxalylglycine (NOG) and m6A-containing ssRNA

Summary for 7WL0

• Entry DOI: 10.2210/pdb7wl0/pdb
• Related: 7V4G 7WKV
• Descriptor: RNA demethylase ALKBH5, RNA (5'-R(*UP*GP*GP*(6MZ)P*CP*UP*GP*C)-3'), MANGANESE (II) ION, ... (6 entities in total)
• Functional Keywords: iron and 2-oxoglutarate dependent oxygenase, rna demethylase, oxidoreductase, double-stranded beta helix, oxidoreductase-rna complex, oxidoreductase/rna
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 10
• Total formula weight: 139533.17

Authors

Kaur, S.,McDonough, M.A.,Schofield, C.J.,Aik, W.S. (deposition date: 2022-01-12, release date: 2022-03-30, Last modification date: 2023-11-29)

Primary citation

Kaur, S.,Tam, N.Y.,McDonough, M.A.,Schofield, C.J.,Aik, W.S.
Mechanisms of substrate recognition and N6-methyladenosine demethylation revealed by crystal structures of ALKBH5-RNA complexes.
Nucleic Acids Res., 50:4148-4160, 2022

PubMed Abstract: AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N6-methyladenosine (m6A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mass and obesity-associated protein (FTO), ALKBH5 is one of only two identified human m6A RNA oxidizing enzymes and is a potential target for cancer treatment. Unlike FTO, ALKBH5 efficiently catalyzes fragmentation of its proposed nascent hemiaminal intermediate to give formaldehyde and a demethylated nucleoside. A detailed analysis of the molecular mechanisms used by ALKBH5 for substrate recognition and m6A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m6A-ssRNA 8-mer substrate and supporting biochemical analyses. Strikingly, the single-stranded RNA substrate binds to the active site of ALKBH5 in a 5'-3' orientation that is opposite to single-stranded or double-stranded DNA substrates observed for other AlkB subfamily members, including single-stranded DNA bound to FTO. The combined structural and biochemical results provide insight into the preference of ALKBH5 for substrates containing a (A/G)m6AC consensus sequence motif. The results support a mechanism involving formation of an m6A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139.

PubMed: 35333330
DOI: 10.1093/nar/gkac195

Experimental method

X-RAY DIFFRACTION (2.5 Å)