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Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation

DOI: 10.1093/nar/gku085 DOI Help
PMID: 24489119 PMID Help

Authors: Wei Shen Aik (University of Oxford) , John Scotti (University of Oxford) , Hwanho Choi (University of Oxford) , Lingzhi Gong (Department of Chemistry, University of Oxford) , Marina Demetriades (Department of Chemistry, University of Oxford) , Christopher J Schofield (Department of Chemistry, University of Oxford) , Michael Mcdonough (Department of Chemistry, University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nucleic Acids Research

State: Published (Approved)
Published: January 2014

Open Access Open Access

Abstract: ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N6-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66–292) to 2.0 Å resolution. ALKBH566–292 has a double-stranded β-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. The active site metal is octahedrally coordinated by an HXD…H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other NAOXs. A large loop (βIV–V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX–nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit.

Journal Keywords: Dioxygenases; Humans; Membrane; Models; Molecular; Protein; RNA; Static Electricity

Subject Areas: Biology and Bio-materials

Instruments: I24-Microfocus Macromolecular Crystallography