Modomics - A Database of RNA Modifications

Published on March 10, 2014 in J Biol Chem volume None.

PubMed ID: 24616105


N6-methylation of adenosine (m6A) 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, 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 to other AlkB proteins, Alkbh5 displays several unique structural features on top of the conserved double-stranded beta-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 Cys230 and Cys267 is key for the selective binding of Alkbh5 to single-stranded RNA/DNA by bringing a flipping motif towards the central beta-helix fold. We generated a substrate-binding model of Alkbh5 based on demethylation activity assay of several structure-guided site-directed mutants. Crystallographic and biochemical studies using various analogs of alpha-ketoglutarate (alpha-KG) 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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