Modomics - A Database of RNA Modifications

Published on None in volume (2020) Cell Res 30: 272-275.

PubMed ID: 32051559

DOI: 10.1038/s41422-019-0233-9


We determined two crystal structures of binary and ternary complex of hALKBH1, hALKBH1-Mn2+ and hALKBH1-Mn2+-α-KG at resolutions of 1.97 and 2.8 Å, respectively. hALKBH1 (referred to as hALKBH1 hereafter) contained the enzymatically active center and retained full demethylation activity toward 6mA ssDNA, but not toward 6mA on dsDNA or m1A on ssDNA, the same as hALKBH1 WT. The overall structures of hALKBH1-Mn2+-α-KG and hALKBH1-Mn2+ are similar, with root mean square deviation of 1.4 Å. The hALKBH1 contains a unique N-terminal Flip0, the nucleotide recognition lid (NRL, containing Flip1 and Flip2), and the central catalytic core. A highly conservative double-stranded β-helix (DSBH) fold in the catalytic core is a characteristic of the α-KG-dependent dioxygenase superfamily. Four antiparallel β-strands β7, β9, β12, and β14 form the major sheet, whereas β8, β10, β11 and β13 form the minor sheet. hALKBH1 has a conservative HxD…H metal ion-binding sequence and an R…R α-KG-binding sequence. Apart from Mn2+, α-KG is further stabilized by three hydrogen bonds formed by the side chains of Asn220, Asn340 and Tyr222 as well as three salt bridges formed by Arg338 and Arg344. Isothermal titration calorimetry analysis of the hALKBH1-α-KG interaction revealed a dissociation constant (Kd) of 4.5 μM and a 1:1 stoichiometry. The total surface area of hALKBH1 decreased obviously from 14,445 to 13,893 Å2 during the addition of α-KG, indicating that hALKBH1 whole structure shrank with the association of α-KG, which caused many conformational changes including those of active sites, β5, β8, Flip1 and Flip2. Moreover, of the eight catalytic residues, Arg344 and Tyr222 showed significant conformational changes, interacting with α-KG through moving 2.5 Å and 2.3 Å toward the active center, respectively. Besides, Tyr184 and Glu236 of Flip2 exhibited the most obvious conformational changes and moved to Arg344, forming a stabilization triangle by hydrogen bonds. The stability triangle of Tyr184-Arg344-Glu236 near the α-KG-binding site is essential for 6mA ssDNA demethylation activity. Any single mutation of the triangle abolished the demethylation activity, but did not reduce the DNA-binding affinity. Interestingly, these large conformational changes induced by α-KG were not observed in other AlkB members, revealing a novel function of the essential triangle as a scaffold for the catalytic activity of hALKBH1.