Crystal Structures of the Human RNA Demethylase Alkbh5 Reveal Basis for Substrate Recognition

Mar 12, 2014The Journal of biological chemistry

Human RNA demethylase Alkbh5 structures show how it recognizes its targets

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Abstract

Five high resolution crystal structures of Alkbh5 reveal unique features that influence its function.

N(6)-Methylation of adenosine is a common modification in mRNA and long non-coding RNA that impacts translation and RNA metabolism.
Alkbh5 and FTO can remove this methyl modification from mRNA.
Alkbh5 has distinct structural features, including a unique 'lid' region that is important for recognizing and catalyzing its substrate.
A disulfide bond between Cys-230 and Cys-267 is essential for Alkbh5's selective binding to single-stranded RNA/DNA.
The active site of Alkbh5 is smaller than that of FTO, allowing it to preferentially bind to small molecule inhibitors.
The findings support the development of selective drugs targeting AlkB family proteins.

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N(6)-Methylation of adenosine is the most ubiquitous and abundant modification of nucleoside in eukaryotic mRNA and long non-coding RNA. This modification plays an essential role in the regulation of mRNA translation and RNA metabolism. Recently, human AlkB homolog 5 (Alkbh5) and fat mass- and obesity-associated protein (FTO) were shown to erase this methyl modification on mRNA. Here, we report five high resolution crystal structures of the catalytic core of Alkbh5 in complex with different ligands. Compared with other AlkB proteins, Alkbh5 displays several unique structural features on top of the conserved double-stranded β-helix fold typical of this protein family. Among the unique features, a distinct "lid" region of Alkbh5 plays a vital role in substrate recognition and catalysis. An unexpected disulfide bond between Cys-230 and Cys-267 is crucial for the selective binding of Alkbh5 to single-stranded RNA/DNA by bringing a "flipping" motif toward the central β-helix fold. We generated a substrate binding model of Alkbh5 based on a demethylation activity assay of several structure-guided site-directed mutants. Crystallographic and biochemical studies using various analogs of α-ketoglutarate revealed that the active site cavity of Alkbh5 is much smaller than that of FTO and preferentially binds small molecule inhibitors. Taken together, our findings provide a structural basis for understanding the substrate recognition specificity of Alkbh5 and offer a foundation for selective drug design against AlkB members.

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Crystal Structures of the Human RNA Demethylase Alkbh5 Reveal Basis for Substrate Recognition

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