The atomic resolution structure of human AlkB homolog 7 (ALKBH7), a key protein for programmed necrosis and fat metabolism

Abstract

ALKBH7 is the mitochondrial AlkB family member that is required for alkylation- and oxidation-induced programmed necrosis. In contrast to the protective role of other AlkB family members after suffering alkylation-induced DNA damage, ALKBH7 triggers the collapse of mitochondrial membrane potential and promotes cell death. Moreover, genetic ablation of mouse Alkbh7 dramatically increases body weight and fat mass. Here, we present crystal structures of human ALKBH7 in complex with Mn(II) and α-ketoglutarate at 1.35 Å or N-oxalylglycine at 2.0 Å resolution. ALKBH7 possesses the conserved double-stranded β-helix fold that coordinates a catalytically active iron by a conserved HX(D/E) … Xn … H motif. Self-hydroxylation of Leu-110 was observed, indicating that ALKBH7 has the potential to catalyze hydroxylation of its substrate. Unlike other AlkB family members whose substrates are DNA or RNA, ALKBH7 is devoid of the "nucleotide recognition lid" which is essential for binding nucleobases, and thus exhibits a solvent-exposed active site; two loops between β-strands β6 and β7 and between β9 and β10 create a special outer wall of the minor β-sheet of the double-stranded β-helix and form a negatively charged groove. These distinct features suggest that ALKBH7 may act on protein substrate rather than nucleic acids. Taken together, our findings provide a structural basis for understanding the distinct function of ALKBH7 in the AlkB family and offer a foundation for drug design in treating cell death-related diseases and metabolic diseases.

Keywords: ALKB; ALKBH7; Crystal Structure; Dioxygenase; Fatty Acid Metabolism; Necrosis (Necrotic Death); Obesity; Protein Hydroxylase; Self-Hydroxylation; X-ray Crystallography.

FIGURE 1.

X-ray diffraction patterns of ALKBH7 and Q90R mutants. A, wild-type ALKBH7 Mn(II)·α-KG complex crystals display a sharp and diffuse diffraction pattern with a lattice-translocation defect. The enlargement is in the left lower panel. B, Q90R mutant of ALKBH7 significantly improves the X-diffraction quality.

FIGURE 2.

Structure-based sequence alignment of ALKBH7 homologs from different species and other AlkB family members. Listed below are the ALKBH7 homolog names followed by GenBank^TM accession number and (% identity) to the human ALKBH7: Homo sapiens gi|14150066| (100%); Macaca mulatta gi|109123098 (97.3%); Canis pulus gi|73987027 (93.7%); Bos taurus gi|114051906 (92.8%); and Mus musculus gi|21313470 (81.9%). Structures used are as follows: ALKBH2 (PDB code 3BUC); ALKBH3 (PDB code 2IUW); ALKBH5 (PDB code 4NRO); ALKBH8 (PDB code 3THT); FTO (PDB code 3LFM); and AlkB (PDB code 3BIE). Secondary structural elements are represented according to the structure of ALKBH7. Residue numbers are labeled according to the sequence of ALKBH7. Residues ligating the catalytic metal ion are colored green, and residues binding α-KG are colored blue. Residues marked with green box indicate those forming Flip1, Flip2, βIV-βV loop, and βVII-βVIII loop of ALKBH2, ALKBH3, ALKBH5, ALKBH8, FTO, and AlkB, the nucleobase or phosphate backbone-binding residues are colored cyan. Residues marked with red box indicate those forming α2-β3 loop, βIV-βV loop, and βVII-βVIII loop of ALKBH7, and the conserved negative residues are colored red.

FIGURE 3.

Overall structure of ALKBH7 in complex with Mn(II) and α-KG. Stereoview of the ALKBH7(17–206) structure in the presence of α-KG and Mn(II). The Mn(II) ion is shown as a red sphere. Key residues as well as α-KG are shown as green sticks. Secondary structures are labeled. The α2-β3 loop and β9-β10 loop are highlighted in red.

FIGURE 4.

Crystallographic trimer of ALKBH7. A, schematic representation of the crystallographic trimer of ALKBH7. The three molecules are shown in green, blue, and yellow. Mn(II) ions at the molecule-molecule interface are shown as red spheres. B, stabilization of Arg-90 by electrostatic interactions and hydrogen bonds with Glu-44 and Arg-91 of the adjacent ALKBH7. C, detailed interaction between neighboring ALKBH7 molecules in the asymmetric unit. Mn(II) is bound by His-65 of one ALKBH7 and Glu-62, Glu-75 of the adjacent ALKBH7. D, detailed interaction between neighboring ALKBH7 molecules in the symmetric unit. Mn(II) ion is chelated by Glu-189 from three molecules.

FIGURE 5.

ALKBH7 has an unusual active center. A, view of the ALKBH7 active site residues, α-KG and Mn(II). F_o − F_c (3.0σ, magenta mesh) is calculated after omitting α-KG and Mn(II). The α-KG and its interacting residues are shown as green sticks. The Mn(II) ion is shown as a red sphere and water is shown as a blue sphere. B, conformation comparison of α-KG and NOG in the two structures. ALKBH7-NOG complex is colored cyan, and ALKBH7-α-KG complex is colored green. F_o − F_c electron density map (blue mesh) of NOG is contoured at 2.0σ. C, equilibrium binding analysis of ALKBH7 with α-KG in the presence of the Mn(II) ion using microscale thermophoresis (MST). D–I, active center of other AlkB family members. AlkB (lime green, PDB code 3BIE), ALKBH2 (blue, PDB code 3BUC), ALKBH3 (pink, PDB code 2IUW), ALKBH5 (magenta, PDB code 4NRO), ALKBH8 (orange, PDB code 3THT), FTO (yellow, PDB code 3LFM), respectively. Residues of the nucleotide recognition lid (NRL) Flip2, which are important in binding nucleobase (white sticks), are colored cyan. Note that Flip2 of ALKBH8 is completely disordered so the residues are not shown. The asparagine that forms a hydrogen bond with α-KG c-1 carboxylate oxygen is highlighted in red. The Mn(II) ion is shown as a red sphere and Fe(II) ion is shown in brown sphere.

FIGURE 6.

ALKBH7 lacks the nucleotide recognition lid, which is essential for binding nucleobase and exhibits a solvent-exposed active site. A, structural superimposition of ALKBH7 and other AlkB family members. The conserved nucleotide recognition lids of AlkB (green), ALKBH2 (blue), ALKBH3 (pink), and ALKBH5 (magenta) are highlighted. The Flip1 and Flip2 of the nucleotide recognition lid are labeled, and the corresponding motifs of ALKBH7 are colored red. Structure comparison clearly reveals the absence of Flip2 in ALKBH7. B, comparison of Flip1 of the nucleic acid oxygenases and the α2-β3 loop of ALKBH7. The conformation of α2-β3 loop is different from that of Flip1 and the α2-β3 loop lacks arginine residues that are important in binding nucleobase and phosphate backbone. C, superimposition of ALKBH7, AlkB-dsDNA (green), and ALKBH2-dsDNA (blue) complexes. The α2-β3 loop of ALKBH7 (red) overlaps with the repaired DNA strands. Note: AlkB and ALKBH2 are not shown. D, comparison of the DSBH fold of AlkB family members. The βIV-βV loop and βVII-βVIII loop are highlighted. E, electrostatic surface representation (basic in blue; acidic in red) of ALKBH7 and ALKBH2. The surface of ALKBH7 in equivalent position with the nucleic acid binding groove of other AlkB family members is negatively charged. The active site of ALKBH7 is solvent-exposed, and the cleft opening into the active site is negatively charged.

FIGURE 7.

Structural comparison of ALKBH7 and PHD2. A, structural superimposition of ALKBH7 and PHD2 in complex with HIF-1α CODD peptide (PDB code 3HQR). ALKBH7, PHD2, and HIF-1α CODD peptides are colored green, pale cyan, and yellow, respectively. The hydroxylated proline and α-KG are shown as sticks. C terminus of PHD2 and α2-β3 loop of ALKBH7 are labeled. B, surface representation of ALKBH7 (green) and the HIF-1α CODD peptide (yellow).

FIGURE 8.

Structure of the hydroxylated leucine in both ALKBH3 and ALKBH7. ALKBH3 and ALKBH7 are colored pink and green, respectively. The α-KG and α-KG c-1 carboxylate coordinating arginine and hydroxylated leucine are shown as sticks. The metal ions are shown as spheres. 2F_o − F_c (contoured at 1σ, in gray) and F_o − F_c (contoured at 3σ, in blue) were calculated after omitting the Leu-110 modification (assigned Oϵ).

FIGURE 9.

MS spectra reveals self-hydroxylation of Leu-110 in ALKBH7. A, MS analysis of the tryptic digest of ALKBH7 showed that the tryptic fragment containing Leu-110 (92–115, 2478.32 Da) appeared as m/z = 1240.17 (unmodified) and 1248.17 (+8) as z = 2+ ions; m/z = 827.12 (unmodified) and 832.45 (+5.33) as z = 3+ ions, demonstrating a +16-Da modification. MS analysis of the tryptic digest of ALKBH7 L110A (B), H121A/H123A (C), and R197A/R203A (D) mutants showed that the fragment 92–115 only appeared at the unmodified mass. Note that the mass of the fragment 92–115 in L110A mutant is 2436.28 Da.