LAUSR

Dear Editor, DNA N-methyladenine (6mA) modification is common in prokaryotes and eukaryotes, involving in gene regulation, transposon, stem cell differentiation, and human tumors. At present, it has been confirmed that 6mA is ubiquitous in the human genome, and [G/C]AGG[C/T] is the most prominent motif for 6mA modification. Human ALKBH1 (hALKBH1), one of the nine human homologs of the AlkB family, is an Fe(II) and αketoglutarate (α-KG)-dependent dioxygenase and highly conserved in mammals. AlkB family proteins can repair damaged DNA/RNA or other lesions. hALKBH1 exhibits demethylation activity toward 6mA, and its abnormal expression has been found in many human cancers and developmental defects, such as tissue malformation and gender imbalance. DNA methyltransferase N6AMT1 and demethylase hALKBH1 mediate the methylation and demethylation of DNA 6mA in the human genome, respectively. The abnormal distribution of 6mA has been found in many cancers. Interestingly, hALKBH1 is also reported to have demethylation activity toward other four kinds of substrates, such as histone H2A, mC on DNA and RNA, mC or mA on tRNA. Therefore, the molecular function of hALKBH1 is still controversial and the functional mechanism is unclear. In addition, the sequence identity between hALKBH1 and other solved structures is less than 19%, which hinders the understanding of action mechanism and the potential drug applications. Here we determined two crystal structures of binary and ternary complex of hALKBH119–369, hALKBH119–369-Mn 2+ and hALKBH119–369-Mn -α-KG at resolutions of 1.97 and 2.8 Å, respectively (Supplementary information, Fig. S1 and Table S1). hALKBH119–369 (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 mA on ssDNA, the same as hALKBH1 WT (Fig. 1a, b). The overall structures of hALKBH1-Mn-α-KG and hALKBH1-Mn are similar, with root mean square deviation of 1.4 Å. The hALKBH1 contains a unique Nterminal 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 (Fig. 1c; Supplementary information, Fig. S2). 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 Mn, 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 (Fig. 1d, e). Isothermal titration calorimetry analysis of the hALKBH1α-KG interaction revealed a dissociation constant (Kd) of 4.5 μM and a 1:1 stoichiometry (Supplementary information, Fig. S3b). The total surface area of hALKBH1 decreased obviously from 14,445 to 13,893 Å 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 (Supplementary information, Movie S1, Fig.S3c, d). 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 (Fig. 1d). 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 (Fig. 1e, f), but did not reduce the DNA-binding affinity (Supplementary information, S3e). 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. Compared with other members of the AlkB family, the NRL of hALKBH1 has several unique structural features (Supplementary information, S4). The hALKBH1 Flip1 region is unique and long, leaving a larger binding space over the active site pocket (Fig. 1g). K158A/R159A/R160A/R162A and K167A/R169A mutants completely lost the 6mA demethylation activity, and the activity of K158A/ R159A/R160A mutant was significantly compromised (Fig.1h). Moreover, the Flip2 contains a pair of antiparallel β-sheets and a long loop with high B factors (Supplementary information, Fig. S5). Structurebased sequence analyses and mutagenesis experiments confirmed that key residues in the NRL region, such as Arg169, Trp170, Tyr177, and Trp179, are potential determinants of hALKBH1 for 6mA recognition and demethylation (Fig. 1i; Supplementary information, Fig. S6). The distinctive composition and conformation of Flip1 and Flip2 are likely to confer substrate selectivity on hALKBH1. Intriguingly, a significant structural feature of hALKBH1 at the Nterminus is the Flip0 (residues 19–32). It is highly conserved in ALKBH1 among various mammalian species, but not in OsALKBH1 or other AlkB family members (Fig. 1j; Supplementary information, Fig. S7). When superimposed with the hALKBH2-dsDNA complex structure, the Flip0 region of hALKBH1 is well accommodated by the modified strand, and the modified nucleotide enters the catalytic pocket. However, Flip0 seriously collides with the unmethylated strand (Fig. 1k). We hypothesized that Flip0 impedes the access of paired dsDNA to the active site and this may be the structural basis for the selectivity of hALKBH1 toward single-stranded substrates. EMSA data revealed that dsDNA and ssDNA can bind to hALKBH1, and EMSA and chromatographic analyses showed a direct binding between Flip0 and ssDNA/dsDNA (Supplementary information, Figs. S8 and S9). However, hALKBH1 has no demethylation activity on 6mA of dsDNA. Based on the above results, we believe that Flip0 is disadvantageous for 6mA dsDNA to enter the hALKBH1 catalytic pocket, and that dsDNA and hALKBH1 are a non-productive combination in the presence of Flip0, rather than a productive way. Flip0 binds tightly adjacent to the minor sheet of the DSBH fold, and most of the interactions are hydrophobic with all the