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

Chong Feng,Yang Liu,Guoqiang Wang,Zengqin Deng,Qi Zhang,Wei Wu,Yufeng Tong,Changmei Cheng,Zhongzhou Chen

doi:10.1074/jbc.m113.546168

Abstract

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.

Highlights

Human AlkB homolog 5 (Alkbh5) is an RNA demethylase that erases m6A modification
We presented five high resolution crystal structures of the Alkbh5 catalytic core without Mn2ϩ, with Mn2ϩ alone, or with Mn2ϩ in addition to another ligands, NOG, ␣-KG, or PDCA
Based on our mutagenesis analyses (Figs. 2C and 5C) and the known substrate recognition mechanisms of other AlkB members [15, 16, 20, 43], the long Flip2 loop of Alkbh5 was proposed to play a significant role in substrate recognition and binding through interaction with one end of the single-stranded nucleic acid substrate (Fig. 5E)

Summary

Background

Human AlkB homolog 5 (Alkbh5) is an RNA demethylase that erases m6A modification. Human AlkB homolog 5 (Alkbh5) and fat mass- and obesity-associated protein (FTO) were shown to erase this methyl modification on mRNA. Given the striking biological role of Alkbh as an m6A RNA demethylase, it is of considerable scientific interest to elucidate the molecular mechanism underlying Alkbh demethylation activity To this end, we determined five crystal structures of the human Alkbh catalytic core in complex with citrate and acetate, Mn2ϩ, and Mn2ϩ with ligand ␣-KG, N-oxalylglycine (NOG), or pyridine 2,4-dicarboxylate (PDCA). Structure comparison with AlkB homologs and activity analysis of site-directed mutants were carried out and provided insights into the distinct substrate preference of Alkbh and selective inhibitor design

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION