Structure of human RNA N⁶-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation.
ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N(6)-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.
Department of Chemistry, Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
WeiShen
W
Aik
John S
JS
Scotti
Hwanho
H
Choi
Lingzhi
L
Gong
Marina
M
Demetriades
Christopher J
CJ
Schofield
Michael A
MA
McDonough
D000071502:AlkB Homolog 5, RNA Demethylase; D020134:Catalytic Domain; D049308:Dioxygenases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D055672:Static Electricity
D008565:Membrane Proteins; D012313:RNA; D049308:Dioxygenases; C559920:ALKBH5 protein, human; D000071502:AlkB Homolog 5, RNA Demethylase