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

Abstract

Background: Human AlkB homolog 5 (Alkbh5) is an RNA demethylase that erases m 6 A modification. Results: Crystal structures of an enzymatically active Alkbh5 construct in complex with cofactors or small molecules were determined. Conclusion: Structure and activity analyses showed that Alkbh5 strongly prefers single-stranded oligos and small molecule inhibitors. Significance: The Alkbh5 structure reveals potential for structure-based design of selective inhibitors.

Structures of Human ALKBH5 Demethylase Reveal a Unique Binding Mode for Specific Single-stranded N6-Methyladenosine RNA Demethylation

“…By molecular modeling of the ALKBH5 substrate complex structure, we identify potential m 6 A binding residues, the identities of which are validated by mutagenesis and ITC binding experiments. During revision of this manuscript, two other manuscripts on human ALKBH5 structures were reported (38,39); their structural data and conclusions are consistent with our structural results, which are presented here.…”

A Radioactivity-Based Assay for Screening Human m6A-RNA Methyltransferase, METTL3-METTL14 Complex, and Demethylase ALKBH5

“…19,26 Briefly, the lysate was passed through a DE52 column at 500 mM NaCl to filter DNA and other nonprotein contaminants followed by the Ni-NTA column. Resin was washed with binding buffer (20 mM Tris, pH 7.5, 500 mM NaCl, 5% glycerol, and 5 mM imidazole) followed by washing with wash buffer (binding buffer with 30 mM imidazole) and eluted with elution buffer (binding buffer with 250 mM imidazole).…”

Update: Mechanisms Underlying N 6 -Methyladenosine Modification of Eukaryotic mRNA

“…Overexpression of FTO or ALKBH5 in cells decreases global m 6 A levels, but knockdown or knockout of either only mildly increases m 6 A levels [25, 66], suggesting the existence of other demethylases or perhaps a synergy between ALKBH5 and FTO that has not been studied. The crystal structures of both FTO and ALKBH5 have been reported, and small molecule inhibitors targeting their demethylase activities have been developed based on these structures [69–71]. For example, the natural product rhein, derived from herbs, is among the most effective FTO m 6 A demethylase inhibitors [72].…”