Structure And Function Studies Of ALKBH7, A Key Protein For Programmed Necrosis

Cell death, one of the most vital cellular responses of multicellular organisms, can be divided into apoptosis, autophagy and necrosis based on different morphological appearances. Although apoptosis is well studied and regarded as programmed cell death, necrosis has been considered as an uncontrolled process that occurs as a result of infection or injury for a long time. However, the accumulating evidences reveal that necrosis is also well-regulated as apoptosis in many cases. For example, the tumor necrosis factor (TNF)-induced programmed necrosis has unique signaling mechanisms. Programmed necrosis plays an important role in various pathological processes, including ischemic brain injury, neurodegenerative diseases and viral infections. Necrosis-mediated via the poly (ADP-ribose) polymerase (PARP) pathway is the other extensively studied model of programmed necrosis. Recently, the mitochondrial protein human AlkB homolog7(ALKBH7) was found to be essential for alkylation-and oxidation-induced programmed necrosis, but it had no effect on apoptosis. In contrast to the protective role of other AlkB family members after suffering alkylation induced DNA damage, ALKBH7triggers the collapse of mitochondrial membrane potential and promotes cell death. Besides, knock-out Alkbh7in mice led to increased body weight and body fat. Alkbh7was found to facilitate the utilization of short chain fatty acids. So far, the structure and activity of ALKBH7remain unclear.Through extensive screening of truncated variants and mutants, ALKBH717-215Q90R protein crystal was obtained. Structures of ALKBH717-206·Mn(Ⅱ)·α-KG and ALKBH717-206·Mn(Ⅱ)-NOG complexes were solved using multiwavelength anomalous diffraction and molecular replacement. A total of ten β-strands surrounded by four a-helices make up the central structure of ALKBH7. The catalytic core of ALKBH7contains a double-stranded β-helix (DSBH) fold conserved in the Fe (Ⅱ)/α-KG-dependent dioxygenase superfamily. The HXD...H...R...R motif of ALKBH7coordinates the metal ion and a-KG at the active site, which is conserved in AlkB family. The a-KG c-1carboxylate oxygen forms a hydrogen bond with asparagine in AlkB family members except ALKBH7. In ALKBH7, a leucine lies in the equivalent position of the above asparagine, leading to weaker binding affinity of a-KG. An extra electron density of Leu110was found in the1.35A ALKBH7·α-KG complex structure, indicating a post-translational modification for Leu110. Self-hydroxylation of Leu110was supported by the results of MS analysis and mutagensis. Observation of self-hydroxylation suggests that ALKBH7could catalyze post-translational modification of its substrates. ALKBH7has a negatively charged surface in equivalent position with the nucleic acid binding groove of other AlkB family members. An additional loop between strands β9and010creates the outer wall of the minor β-sheet of DSBH, four acidic residues in this loop are responsible for the negatively charged surface of ALKBH7. The conserved nucleotide recognition lid of AlkB family members is absent in ALKBH7. Without the essential nucleotide recognition lid, ALKBH7displays a solvent-exposed active site incapable of positioning the modified nucleobase. Binding of nucleic acid would be impossible because of the charge repulsion between the phosphate backbone and ALKBH7. These features basically rules out the possibility that ALKBH7functions as a nucleic acid oxygenase. Structural comparison of ALKBH7and other Fe(Ⅱ)and a-KG dependent dioxygenases reveals the potential role of ALKBH7as a protein hydroxylase. ALKBH7has a negatively charged cleft opening into the active site. Proline and lysine/arginine-containing proteins or peptides, which could be the substrates of ALKBH7,need to be further identified.Taken together, this work provides a structural basis for understanding the unique role of ALKBH7distinct from the known ALKBH functions such as DNA repair or RNA modification. The results point the way for future studies to address the signaling mechanism of the alkylation-and oxidation-induced programmed necrosis. The structure of ALKBH7can also be exploited for the development of selective inhibitors to avoid unwarranted cell death in wounded or patients.