Structural insights into the interactions and epigenetic functions of human nucleic acid repair protein ALKBH6
2022; Elsevier BV; Volume: 298; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2022.101671
ISSN1083-351X
AutoresLulu Ma, Hongyun Lu, Zizi Tian, Meiting Yang, Jun Ma, Guohui Shang, Yunlong Liu, Mengjia Xie, Guoguo Wang, Wei Wu, Ziding Zhang, Shaodong Dai, Zhongzhou Chen,
Tópico(s)Biochemical and Molecular Research
ResumoHuman AlkB homolog 6, ALKBH6, plays key roles in nucleic acid damage repair and tumor therapy. However, no precise structural and functional information are available for this protein. In this study, we determined atomic resolution crystal structures of human holo-ALKBH6 and its complex with ligands. AlkB members bind nucleic acids by NRLs (nucleotide recognition lids, also called Flips), which can recognize DNA/RNA and flip methylated lesions. We found that ALKBH6 has unusual Flip1 and Flip2 domains, distinct from other AlkB family members both in sequence and conformation. Moreover, we show that its unique Flip3 domain has multiple unreported functions, such as discriminating against double-stranded nucleic acids, blocking the active center, binding other proteins, and in suppressing tumor growth. Structural analyses and substrate screening reveal how ALKBH6 discriminates between different types of nucleic acids and may also function as a nucleic acid demethylase. Structure-based interacting partner screening not only uncovered an unidentified interaction of transcription repressor ZMYND11 and ALKBH6 in tumor suppression but also revealed cross talk between histone modification and nucleic acid modification in epigenetic regulation. Taken together, these results shed light on the molecular mechanism underlying ALKBH6-associated nucleic acid damage repair and tumor therapy. Human AlkB homolog 6, ALKBH6, plays key roles in nucleic acid damage repair and tumor therapy. However, no precise structural and functional information are available for this protein. In this study, we determined atomic resolution crystal structures of human holo-ALKBH6 and its complex with ligands. AlkB members bind nucleic acids by NRLs (nucleotide recognition lids, also called Flips), which can recognize DNA/RNA and flip methylated lesions. We found that ALKBH6 has unusual Flip1 and Flip2 domains, distinct from other AlkB family members both in sequence and conformation. Moreover, we show that its unique Flip3 domain has multiple unreported functions, such as discriminating against double-stranded nucleic acids, blocking the active center, binding other proteins, and in suppressing tumor growth. Structural analyses and substrate screening reveal how ALKBH6 discriminates between different types of nucleic acids and may also function as a nucleic acid demethylase. Structure-based interacting partner screening not only uncovered an unidentified interaction of transcription repressor ZMYND11 and ALKBH6 in tumor suppression but also revealed cross talk between histone modification and nucleic acid modification in epigenetic regulation. Taken together, these results shed light on the molecular mechanism underlying ALKBH6-associated nucleic acid damage repair and tumor therapy. As α-ketoglutarate (α-KG) and Fe(II)-dependent dioxygenases, AlkB family members are widespread in the biological kingdom and play important roles in epigenetics. The AlkB gene was found in Escherichia coli, and its product was involved in repairing alkylated bases (1Kataoka H. Yamamoto Y. Sekiguchi M. A new gene (alkB) of Escherichia coli that controls sensitivity to methyl methane sulfonate.J. Bacteriol. 1983; 153: 1301-1307Google Scholar). Human genome encodes nine AlkB homologs named ALKBH1–8 and FTO (fat mass and obesity-associated) with low sequence identity (2Kurowski M.A. Bhagwat A.S. Papaj G. Bujnicki J.M. Phylogenomic identification of five new human homologs of the DNA repair enzyme AlkB.BMC Genomics. 2003; 4: 48Google Scholar, 3Gerken T. Girard C.A. Tung Y.C. Webby C.J. Saudek V. Hewitson K.S. Yeo G.S. McDonough M.A. Cunliffe S. McNeill L.A. Galvanovskis J. Rorsman P. Robins P. Prieur X. Coll A.P. et al.The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.Science. 2007; 318: 1469-1472Google Scholar). All the nine AlkB homologs have different substrates and functions including DNA repair, RNA modification, actomyosin demethylation, and fatty acid metabolism (4Falnes P.O. Repair of 3-methylthymine and 1-methylguanine lesions by bacterial and human AlkB proteins.Nucleic Acids Res. 2004; 32: 6260-6267Google Scholar, 5Lee D.H. Jin S.G. Cai S. Chen Y. Pfeifer G.P. O'Connor T.R. Repair of methylation damage in DNA and RNA by mammalian AlkB homologues.J. Biol. Chem. 2005; 280: 39448-39459Google Scholar, 6Li M.-M. Nilsen A. Shi Y. Fusser M. Ding Y.-H. Fu Y. Liu B. Niu Y. Wu Y.-S. Huang C.-M. Olofsson M. Jin K.-X. Lv Y. Xu X.-Z. He C. et al.ALKBH4-dependent demethylation of actin regulates actomyosin dynamics.Nat. Commun. 2013; 4: 1832Google Scholar). Most of them have essential roles in metabolism and diseases curbing such as cancers (7Xie Q. Wu T.P. Gimple R.C. Li Z. Prager B.C. Wu Q. Yu Y. Wang P. Wang Y. Gorkin D.U. Zhang C. Dowiak A.V. Lin K. Zeng C. Sui Y. et al.N(6)-methyladenine DNA modification in glioblastoma.Cell. 2018; 175: 1228-1243.e20Google Scholar, 8Xiao C.L. Zhu S. He M. Chen D. Zhang Q. Chen Y. Yu G. Liu J. Xie S.Q. Luo F. Liang Z. Wang D.P. Bo X.C. Gu X.F. Wang K. et al.N(6)-Methyladenine DNA modification in the human genome.Mol. Cell. 2018; 71: 306-318.e7Google Scholar, 9Ruan D.Y. Li T. Wang Y.N. Meng Q. Li Y. Yu K. Wang M. Lin J.F. Luo L.Z. Wang D.S. Lin J.Z. Bai L. Liu Z.X. Zhao Q. Wu X.Y. et al.FTO downregulation mediated by hypoxia facilitates colorectal cancer metastasis.Oncogene. 2021; 40: 5168-5181Google Scholar). Currently, more than 20 kinds of nucleic acids methylated adducts have been reported as AlkB members substrates (10Perry G.S. Das M. Woon E.C.Y. Inhibition of AlkB nucleic acid demethylases: Promising new epigenetic targets.J. Med. Chem. 2021; 64: 16974-17003Google Scholar). The AlkB family members have broad substrates selectivity: ALKBH1 can demethylate DNA 6mA modification enriched in human glioblastoma (7Xie Q. Wu T.P. Gimple R.C. Li Z. Prager B.C. Wu Q. Yu Y. Wang P. Wang Y. Gorkin D.U. Zhang C. Dowiak A.V. Lin K. Zeng C. Sui Y. et al.N(6)-methyladenine DNA modification in glioblastoma.Cell. 2018; 175: 1228-1243.e20Google Scholar); it also can demethylate 3mC, 1mA, et al. (11Westbye M.P. Feyzi E. Aas P.A. Vagbo C.B. Talstad V.A. Kavli B. Hagen L. Sundheim O. Akbari M. Liabakk N.B. Slupphaug G. Otterlei M. Krokan H.E. Human AlkB homolog 1 is a mitochondrial protein that demethylates 3-methylcytosine in DNA and RNA.J. Biol. Chem. 2008; 283: 25046-25056Google Scholar, 12Liu F. Clark W. Luo G. Wang X. Fu Y. Wei J. Wang X. Hao Z. Dai Q. Zheng G. Ma H. Han D. Evans M. Klungland A. Pan T. et al.ALKBH1-mediated tRNA demethylation regulates translation.Cell. 2016; 167: 816-828.e16Google Scholar). ALKBH2 demethylates 1mA and 3mC both in ssDNA and dsDNA; ALKBH3 preferentially demethylates 1mA and 3mC in ssDNA (13Duncan T. Trewick S.C. Koivisto P. Bates P.A. Lindahl T. Sedgwick B. Reversal of DNA alkylation damage by two human dioxygenases.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16660-16665Google Scholar). ALKBH4, without any structural information, can demethylate 6mA on dsDNA and might also on actin K84me1 (6Li M.-M. Nilsen A. Shi Y. Fusser M. Ding Y.-H. Fu Y. Liu B. Niu Y. Wu Y.-S. Huang C.-M. Olofsson M. Jin K.-X. Lv Y. Xu X.-Z. He C. et al.ALKBH4-dependent demethylation of actin regulates actomyosin dynamics.Nat. Commun. 2013; 4: 1832Google Scholar). ALKBH5 and ALKBH8 can demethylate m6A RNA or mcm5U RNA, respectively (10Perry G.S. Das M. Woon E.C.Y. Inhibition of AlkB nucleic acid demethylases: Promising new epigenetic targets.J. Med. Chem. 2021; 64: 16974-17003Google Scholar). ALKBH7 is recently reported to be able to demethylate m22G and m1A of mt-RNA (14Zhang L.S. Xiong Q.P. Pena Perez S. Liu C. Wei J. Le C. Zhang L. Harada B.T. Dai Q. Feng X. Hao Z. Wang Y. Dong X. Hu L. Wang E.D. et al.ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing.Nat. Cell Biol. 2021; 23: 684-691Google Scholar). FTO prefers to catalyze RNA substrates; it can suppress colorectal cancer with a better prognosis in an m6A-dependent manner, which highlights its clinical functions (9Ruan D.Y. Li T. Wang Y.N. Meng Q. Li Y. Yu K. Wang M. Lin J.F. Luo L.Z. Wang D.S. Lin J.Z. Bai L. Liu Z.X. Zhao Q. Wu X.Y. et al.FTO downregulation mediated by hypoxia facilitates colorectal cancer metastasis.Oncogene. 2021; 40: 5168-5181Google Scholar). Besides, we previously solved structures of human ALKBH5, ALKBH7, and ALKBH1 (15Feng C. Liu Y. Wang G. Deng Z. Zhang Q. Wu W. Tong Y. Cheng C. Chen Z. Crystal structures of the human RNA demethylase Alkbh5 reveal basis for substrate recognition.J. Biol. Chem. 2014; 289: 11571-11583Google Scholar, 16Wang G. He Q. Feng C. Liu Y. Deng Z. Qi X. Wu W. Mei P. Chen Z. The atomic resolution structure of human AlkB homolog 7 (ALKBH7), a key protein for programmed necrosis and fat metabolism.J. Biol. Chem. 2014; 289: 27924-27936Google Scholar, 17Tian L.F. Liu Y.P. Chen L. Tang Q. Wu W. Sun W. Chen Z. Yan X.X. Structural basis of nucleic acid recognition and 6mA demethylation by human ALKBH1.Cell Res. 2020; 30: 272-275Google Scholar), providing precise structural information for function research. Being a member of the AlkB family, ALKBH6 located in nuclei and cytoplasm is widely distributed with the highest expression in testis and pancreas (2Kurowski M.A. Bhagwat A.S. Papaj G. Bujnicki J.M. Phylogenomic identification of five new human homologs of the DNA repair enzyme AlkB.BMC Genomics. 2003; 4: 48Google Scholar, 18Tsujikawa K. Koike K. Kitae K. Shinkawa A. Arima H. Suzuki T. Tsuchiya M. Makino Y. Furukawa T. Konishi N. Yamamoto H. Expression and sub-cellular localization of human ABH family molecules.J. Cell. Mol. Med. 2007; 11: 1105-1116Google Scholar). Chromosomal gain of ALKBH6 is frequently detected in embryonal rhabdomyosarcoma but not in alveolar rhabdomyosarcoma, suggesting that ALKBH6 may play important roles in embryonal rhabdomyosarcoma (19Liu C. Li D. Hu J. Jiang J. Zhang W. Chen Y. Cui X. Qi Y. Zou H. Zhang W. Li F. Chromosomal and genetic imbalances in Chinese patients with rhabdomyosarcoma detected by high-resolution array comparative genomic hybridization.Int. J. Clin. Exp. Pathol. 2014; 7: 690-698Google Scholar). Besides, mice immunized with a mutant peptide of ALKBH6 can suppress tumor growth significantly, whereas the wildtype cannot (20Duan F. Duitama J. Al Seesi S. Ayres C.M. Corcelli S.A. Pawashe A.P. Blanchard T. McMahon D. Sidney J. Sette A. Baker B.M. Mandoiu I.I. Srivastava P.K. Genomic and bioinformatic profiling of mutational neoepitopes reveals new rules to predict anticancer immunogenicity.J. Exp. Med. 2014; 211: 2231-2248Google Scholar). ALKBH6 has been identified as a "cancer gene" in endometrial tumors (21Lawrence M.S. Stojanov P. Mermel C.H. Robinson J.T. Garraway L.A. Golub T.R. Meyerson M. Gabriel S.B. Lander E.S. Getz G. Discovery and saturation analysis of cancer genes across 21 tumour types.Nature. 2014; 505: 495-501Google Scholar). Recently, researchers showed that ALKBH6 can complement AlkB-deficient E. coli strain resistance against alkylating agent methyl methane sulfonate (22Zhao S. Devega R. Francois A. Kidane D. Human ALKBH6 is required for maintenance of genomic stability and promoting cell survival during exposure of alkylating agents in pancreatic cancer.Front. Genet. 2021; 12: 635808Google Scholar). During exposure to the alkylating agent, ALKBH6 plays a crucial role in protecting pancreatic cancer from alkylating-induced DNA damage and promoting cell survival. Furthermore, the overexpression of ALKBH6 in patients with pancreatic cancer has been linked to better survival outcomes (22Zhao S. Devega R. Francois A. Kidane D. Human ALKBH6 is required for maintenance of genomic stability and promoting cell survival during exposure of alkylating agents in pancreatic cancer.Front. Genet. 2021; 12: 635808Google Scholar). The above information indicates that ALKBH6 may play key roles in DNA damage repair and tumor therapy. Huong et al. (23Huong T.T. Ngoc L.N.T. Kang H. Functional characterization of a putative RNA demethylase ALKBH6 in Arabidopsis growth and abiotic stress responses.Int. J. Mol. Sci. 2020; 21: 6707Google Scholar) reported that Arabidopsis ALKBH6, with 36% identity to human ALKBH6, could affect seed germination and survival under abiotic stress serving as a potential eraser protein of RNA methylation. Besides, when rice faced drought, cold, or ABA treatment, the expression level of ALKBH6 was decreased. These data suggest that plant ALKBH6 might be instrumental in abiotic stress responses (24Hu J. Manduzio S. Kang H. Epitranscriptomic RNA methylation in plant development and abiotic stress responses.Front. Plant Sci. 2019; 10: 500Google Scholar). However, the structure, interacting partner, substrates, and activity of human ALKBH6 remain fantasy to date. Moreover, ALKBH6 has a low sequence identity (less than 18.5%) to all the released structures, hindering further research of its function. In this article, we present two high-resolution structures of human ALKBH6, in complex with Tris or cofactor α-KG. These structures reveal a conventional β-strand jelly-roll fold with multiple unique features. With multiple kinds of nucleic acids screened, we demonstrated that ALKBH6 could bind nucleic acids and it preferred single-stranded nucleic acids. Structure-based interacting partner screening found that ALKBH6 could physically interact with both itself and ZMYND11, which offered us novel clues for functional research. The human ALKBH6 protein expressed from E. coli failed to form crystal after extensive screening both fragments and crystallization conditions. By using the Pichia pastoris X-33 expression system, we successfully got the full-length ALKBH6 protein and performed crystal screening. Finally, high-resolution crystals of ALKBH6 in complex with Tris and cofactor α-KG were obtained after optimization. However, owing to the low sequence homology (less than 18.5%) to all structures in the Protein Data Bank (PDB), all molecular replacements failed when using single or multiple homologous structures as models. Then we tried different truncations of the predicted structure by Raptorx (25Kallberg M. Wang H. Wang S. Peng J. Wang Z. Lu H. Xu J. Template-based protein structure modeling using the RaptorX web server.Nat. Protoc. 2012; 7: 1511-1522Google Scholar) as molecular replacement (MR) models, such as removing disordered loops or short β-sheets. After substantial trials of different resolution and space groups, the best solution was found by BALBES (26Long F. Vagin A.A. Young P. Murshudov G.N. BALBES: A molecular-replacement pipeline.Acta Crystallogr. D Biol. Crystallogr. 2008; 64: 125-132Google Scholar). After numerous cycles of manual rebuilding and refining, the final structure of the ALKBH6∙Ni2+∙Tris was refined to a resolution of 1.79 Å in the space group P212121, with an Rwork of 19.8% and an Rfree of 21.8% (Table 1). The structure of ALKBH6 complexed with cofactor α-KG and Mn2+ (holo-ALKBH6) was determined by molecular replacement using the ALKBH6∙Ni2+∙Tris structure as a model and was refined to a final Rwork of 17.2% and Rfree of 19.7% at 1.75 Å (Table 1). Each cell contains one ALKBH6 molecule (Fig. S1A). An overlay of holo-ALKBH6 and ALKBH6∙Ni2+∙Tris structures revealed high similarity in the overall conformation with a root-mean-square deviation (r.m.s.d.) of 0.2 Å (Fig. S1A).Table 1Data collection and refinement statistics of ALKBH6 complexesDataALKBH6∙Ni2+∙TrisALKBH6∙Mn2+∙α-KGData collection Space groupP212121P212121 Cell dimensionsa,b,c (Å)46.4, 64.2, 89.046.1, 64.4, 88.4α,β,γ (˚)90, 90, 9090, 90, 90 Resolution (Å)aStatistics for highest resolution shell.50.0–1.79 (1.84–1.79)50.0–1.75 (1.78–1.75) Rmerge9.0% (79.0%)9.9% (46.9%) I/σ31.5 (2.4)15.0 (2.1) Completeness (%)99.8 (87.1)99.4(95.1) Total no. of reflections180,599122,192 Unique reflections25,64327,487 Redundancy7.1 (3.3)4.5 (3.5)Refinement Resolution (Å)50.0–1.79 (1.84–1.79)50.0–1.75 (1.80–1.75) No. of reflections24,11125,773 Rwork/Rfree (%)19.8/21.817.2/19.7 No. of atomsProtein15691706Ligand/ions911Water8986 Average B-factors (ÅbResidues in favored, allowed, and outlier regions of the Ramachandran plot, respectively.)Protein24.622.2Ligand/ion25.316.9Water29.123.6 rms deviationsBond lengths (Å)0.0080.009Bond angles (º)1.4671.495 Ramachandran plot (%)bResidues in favored, allowed, and outlier regions of the Ramachandran plot, respectively.97.5/2.5/099.5/0.5/0 Molprobity scoreOverall1.26 (98th percentile)1.01 (100th percentile)Clashcore1.91 (100th percentile)2.07 (99th percentile)a Statistics for highest resolution shell.b Residues in favored, allowed, and outlier regions of the Ramachandran plot, respectively. Open table in a new tab The overall structure of ALKBH6 shows a double-stranded β-helix (DSBH) fold at the catalytic core, which is conserved in the Fe(II)/α-KG-dependent dioxygenase superfamily (27McDonough M.A. Loenarz C. Chowdhury R. Clifton I.J. Schofield C.J. Structural studies on human 2-oxoglutarate dependent oxygenases.Curr. Opin. Struct. Biol. 2010; 20: 659-672Google Scholar). The overall similarity is evident in the central DSBH core. DALI search (http://www2.ebi.ac.uk/dali) reveals that S. Pombe OFD2 (PDB: 5YL6) is the most similar structural homolog of the ALKBH6 in the PDB database, with the Z-score of 19.2 and an r.m.s.d. of 2.4 Å over 164 Cα atoms. The ALKBH6 structure also has values comparable with other solved human AlkB family members, such as ALKBH8 (the Z-score of 19.0 and r.m.s.d. of 2.2 Å), ALKBH5 (the Z-score of 18.5 and r.m.s.d. of 2.5 Å), and ALKBH3 (the Z-score of 18.4 and r.m.s.d. of 2.1 Å). Significant differences occur in the secondary structures out of the DSBH fold, such as the protein-interacting regions and the substrate-binding regions near the active sites (Fig. S1B). In ALKBH6, 11 antiparallel β-strands constitute the DSBH domain, which is surrounded by seven α-helices. The DSBH contains a major sheet formed by seven β-strands (β1, β2, β3, β4, β6, β9, and β13), a minor sheet formed by four β-strands (β5, β7, β8, and β10), and sandwiches Mn2+ and α-KG at the active center (Fig. 1A). Two large α-helices (α3 and α5) and three small α-helices (α1, α2, and α4) buttress the major sheet. Consistent with other AlkB family members, ALKBH6 also has Flip1 (residues from A45 to R56) and Flip2 (residues from G62 to P76) (Fig. 1). Interesting, a unique loop (residues from E139 to P158) including a 310 helix (α6) inserts between β7 and β8 in the ALKBH6∙Mn2+∙α-KG structure, nearly overhanging vertical to DSBH, hereafter named Flip3 (Fig. 1). However, in the ALKBH6∙Ni2+∙Tris structure, most of the Flip3 (residues from P140 to P155) had no visible electron density and, therefore, could not be built (Fig. S1A). The reason may be that the cofactor α-KG from the symmetric unit induces the stabilization of the Flip3 conformation in the crystal packing. Meanwhile, residues from A195 to G210 between short β11 and β12 had no visible electron density in either ALKBH6∙Ni2+∙Tris or ALKBH6∙Mn2+∙α-KG structures and thus could not be built. At the same time, these two short β-sheets (β11 and β12) were far away from the potential active sites with high temperature factors (Fig. S1C) and they had an almost 90-degree bend conformation. These two β-sheets and the disorder region between them might mediate regulatory interactions with the DSBH minor sheet as in the ALKBH8. In a previous study (27McDonough M.A. Loenarz C. Chowdhury R. Clifton I.J. Schofield C.J. Structural studies on human 2-oxoglutarate dependent oxygenases.Curr. Opin. Struct. Biol. 2010; 20: 659-672Google Scholar), the H × D…H motif is conserved in the active center of the Fe(II)/α-KG-dependent dioxygenase superfamily. Here in ALKBH6, H114, D116, and H182 compose the motif of H × D…H and coordinate the metal ion stably. In the active center of the apo-ALKBH6 (ALKBH6∙Ni2+∙Tris) structure, an electron density of Tris, from the buffer, was found unexpectedly, which had not been reported in other AlkB family members (Fig. 2A). Surprising, Tris tridentately chelates Ni2+, which forms hexadentate coordination and forms two hydrogen bonds with N103 and D116 of ALKBH6. In the ALKBH6∙Mn2+∙α-KG complex, cofactor α-KG coordinates the Mn2+ bidentately in a standard conformation resembling that seen in other iron(II)/α-KG-based oxygenases and binds to the side chains of N103, Y105, R218, and S220 from β4 or β13 by hydrogen bonds or salt bridges (Fig. 2B). Thus, there are fewer interactions between Tris and ALKBH6 compared with those in the ALKBH6∙Mn2+∙α-KG complex. Besides, the key residues in the above interaction network have no evident conformational difference between the ALKBH6∙Ni2+∙Tris and ALKBH6∙Mn2+∙α-KG structures. Consistently, the defective mutant of three residues (mutant A, N103A/Y105A/R218A) can sharply reduce the binding of α-KG to ALKBH6 (Fig. 2C). Although ALKBH4 was reported to have protein substrates (6Li M.-M. Nilsen A. Shi Y. Fusser M. Ding Y.-H. Fu Y. Liu B. Niu Y. Wu Y.-S. Huang C.-M. Olofsson M. Jin K.-X. Lv Y. Xu X.-Z. He C. et al.ALKBH4-dependent demethylation of actin regulates actomyosin dynamics.Nat. Commun. 2013; 4: 1832Google Scholar), all structure-solved human AlkB family members had nucleic acid oxygenase activity. ALKBH7 was recently reported to be able to demethylate m22G and m1A of mt-RNA (14Zhang L.S. Xiong Q.P. Pena Perez S. Liu C. Wei J. Le C. Zhang L. Harada B.T. Dai Q. Feng X. Hao Z. Wang Y. Dong X. Hu L. Wang E.D. et al.ALKBH7-mediated demethylation regulates mitochondrial polycistronic RNA processing.Nat. Cell Biol. 2021; 23: 684-691Google Scholar). However, the substrate(s) of ALKBH6 remains a mystery for decades. Structural analyses of secondary structures out of the DSBH domain might give some clues. To characterize the difference between ALKBH6 and other AlkB proteins, we overlayed their structures. AlkB family proteins bind and immobilize substrates by nucleotide recognition lids (NRLs, also called Flips), containing several key loops around the catalytic domain (28Xu B. Liu D. Wang Z. Tian R. Zuo Y. Multi-substrate selectivity based on key loops and non-homologous domains: New insight into ALKBH family.Cell. Mol. Life Sci. 2020; 78: 129-141Google Scholar). The NRLs contribute to substrates' selectivity (28Xu B. Liu D. Wang Z. Tian R. Zuo Y. Multi-substrate selectivity based on key loops and non-homologous domains: New insight into ALKBH family.Cell. Mol. Life Sci. 2020; 78: 129-141Google Scholar, 29Sundheim O. Talstad V.A. Vågbø C.B. Slupphaug G. Krokan H.E. AlkB demethylases flip out in different ways.DNA Repair. 2008; 7: 1916-1923Google Scholar) and are less conserved among AlkB members. The Flip1 and Flip2 of ALKBH6 (Fig. 3A) form the NRL domain and are notably different from those of other human AlkB family proteins (Figs. 3B and S1B). The Flip1 of ALKBH6 contains a short α-helix α4, a short β-sheet β2, and a loop (Fig. 3B). It is the shortest one among the family members, which may affect substrates' binding. The β2 is the outermost β-sheet of the seven-stranded major sheet and is less conserved in the family. Owing to the high temperature factors of Flip1 (Fig. S1C), it may take a conformational change upon substrate binding. In the ALKBH2–dsDNA complex structure, F102, which locates in the Flip1 of ALKBH2, intercalates into the duplex stack and fills the DNA gap (30Yang C.G. Yi C. Duguid E.M. Sullivan C.T. Jian X. Rice P.A. He C. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA.Nature. 2008; 452: 961-965Google Scholar), whereas ALKBH6 lacks any aromatic residues in the corresponding position (Fig. S2A). The Flip2 of ALKBH6 is solved as a loop (Fig. 3A). Surprising, among the human AlkB family proteins, only the Flip2 of ALKBH6 does not have any aromatic residue (Fig. S2A). In the total 27 residues of Flip1 and Flip2, only 1 aromatic residue W50 exists. Of note, Flip1 and Flip2 of ALKBH6 have a higher basic amino acids ratio (7/27) compared with ALKBH2 (5/40), ALKBH3 (6/35), or ALKBH5 (6/45). Moreover, a prominent feature of the NRL domain is that Flip2 is restrained by the C-terminal loop (hereafter Loop-CTD, residues V226–K238). Flip2 has hydrophobic interaction network with Loop-CTD. Especially, the ALKBH6 Loop-CTD has the highest content of hydrophobic residues (7/13) among the family (Fig. S2A). Consistently, an extensive hydrophobic network is formed by P65, M70, V71, P72 from Flip2 and V226, V229, L230, L235 from Loop-CTD. Moreover, the main chain of residue M70 forms two hydrogen bonds with main chains of residues R228 and L230 from Loop-CTD. The side chain of R68 forms two hydrogen bonds with the carbonyl group of residue G237 (Fig. S2B). Thus, Loop-CTD greatly stabilizes the conformation of Flip2 and the average temperature factor of Flip2 is lower than that of Flip1 (Fig. S1C). As the substrate for ALKBH6 is still unknown, we carefully analyze the structure and superpose with homologous members. The distribution of the positive charge of ALKBH6 is different from that of the other family proteins (Fig. S2C). ALKBH6 has fewer positively charged residues compared with ALKBH1 or ALKBH2 at the surface of the active site. But it has some similarities to ALKBH3 and ALKBH5. A positively charged groove traverses the potential active site between Flip1 and Flip2; however, it is very narrow. The positively charged groove may directly bind the substrates. Most of the structure-solved AlkB family members have dynamic flexible Flips to dock substrates into the active site. However, it is difficult to accurately figure out the natural substrates of ALKBH6 because of these characteristic flexible Flips. As the residues in the DSBH core shift less when binding nucleotides, here we analyze some of these residues involved in nucleotides' recognition. In the structure of the ALKBH2-dsDNA complex (30Yang C.G. Yi C. Duguid E.M. Sullivan C.T. Jian X. Rice P.A. He C. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA.Nature. 2008; 452: 961-965Google Scholar), the nucleobase ring of cytidine forms aromatic stacking interaction with the Fe2+-ligating residue H171, while H114 is in the same conserved conformation in ALKBH6. In structures of the human AlkB family members, the C terminus of Flip1 (except in ALKBH7) has a key positively charged residue that is involved in interacting with the nucleic acid (Fig. S2, A and D). R110 in ALKBH2 directly binds the phosphate backbone (30Yang C.G. Yi C. Duguid E.M. Sullivan C.T. Jian X. Rice P.A. He C. Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA.Nature. 2008; 452: 961-965Google Scholar); ALKBH6 R56 and ALKBH8 R174 are in similar positions (31Pastore C. Topalidou I. Forouhar F. Yan A.C. Levy M. Hunt J.F. Crystal structure and RNA binding properties of the RNA recognition motif (RRM) and AlkB domains in human AlkB homolog 8 (ABH8), an enzyme catalyzing tRNA hypermodification.J. Biol. Chem. 2012; 287: 2130-2143Google Scholar). In addition, the positions of I111 and R224 are invariant among ALKBH2, ALKBH6, and ALKBH8. Moreover, the ALKBH6 active center contains four residues (L58, N60, L101, and Y120) that are similar to those in ALKBH2 (Fig. S2D). Therefore, these similarities reveal that ALKBH6 might act as a DNA/RNA demethylase like other human AlkB family members. As reported, expression of ALKBH6 in AlkB-deficient E. coli strain increases survival dramatically after methyl methane sulfonate treatment (22Zhao S. Devega R. Francois A. Kidane D. Human ALKBH6 is required for maintenance of genomic stability and promoting cell survival during exposure of alkylating agents in pancreatic cancer.Front. Genet. 2021; 12: 635808Google Scholar). Based on the structural clues, we proposed that ALKBH6 could interact with nucleic acids directly. Then we explored various substrates, such as dsDNA, ssDNA, RNA, and bubble or bulge DNA with different numbers of mismatched base pairs in the middle of dsDNA (17Tian L.F. Liu Y.P. Chen L. Tang Q. Wu W. Sun W. Chen Z. Yan X.X. Structural basis of nucleic acid recognition and 6mA demethylation by human ALKBH1.Cell Res. 2020; 30: 272-275Google Scholar, 32Zhang M. Yang S. Nelakanti R. Zhao W. Liu G. Li Z. Liu X. Wu T. Xiao A. Li H. Mammalian ALKBH1 serves as an N6-mA demethylase of unpairing DNA.Cell Res. 2020; 30: 197-210Google Scholar). With extensive and multiple screening, we finally found that ALKBH6 could bind ssDNA (Fig. 3, C and D and Table S1), bubble DNA (Figs. 3E and S3A), bulge DNA (Fig. S3B), and RNA (Fig. S3, C and D) but not dsDNA (Fig. S3E). All the nucleic acids have no notable conservative motif or any characteristics such as GC rich or AT rich. To eliminate the nonspecific binding, an unlabeled ssDNA1 competitor was used to rule out spurious interaction. With the competitor concentration increasing, the FAM-labeled complex signal decreased (Fig. 3D). In Arabidopsis, ALKBH6 binds to m6A and m5C RNA (23Huong T.T. Ngoc L.N.T. Kang H. Functional characterization of a putative RNA demethylase ALKBH6 in Arabidopsis growth and abiotic stress responses.Int. J. Mol. Sci. 2020; 21: 6707Google Scholar), but the enzymatic activity has not been reported. Human ALKBH6 has a 36% identity with its homology in Arabidopsis. It implies that human ALKBH6 might bind RNA. We used FAM-labeled 30-nt poly(A) as a potential substrate to incubate with ALKBH6. We got a similar binding affinity as ssDNA by microscale thermophoresis (MST) and a complex band in EMSA (Fig. S3C). In eukaryotes, the mRNA has a 3′-poly(A); the above results prompt us to think if ALKBH6 could bind to mRNA and this awaits further demonstration. When the mutant ALKBH6ΔLoop-CTD is used to test the binding with bubble or bulge DNA, it shows a compromised binding affinity (Figs. S3A and 3B). Therefore, the Loop-CTD has a moderately positive effect on binding bubble or bulge DNA. To know the key re
Referência(s)