Crystal Structures of the Human RNA Demethylase Alkbh5 Reveal Basis for Substrate Recognition*

Background: Human AlkB homolog 5 (Alkbh5) is an RNA demethylase that erases m6A modification. Results: Crystal structures of an enzymatically active Alkbh5 construct in complex with cofactors or small molecules were determined. Conclusion: Structure and activity analyses showed that Alkbh5 strongly prefers single-stranded oligos and small molecule inhibitors. Significance: The Alkbh5 structure reveals potential for structure-based design of selective inhibitors. N6-Methylation of adenosine is the most ubiquitous and abundant modification of nucleoside in eukaryotic mRNA and long non-coding RNA. This modification plays an essential role in the regulation of mRNA translation and RNA metabolism. Recently, human AlkB homolog 5 (Alkbh5) and fat mass- and obesity-associated protein (FTO) were shown to erase this methyl modification on mRNA. Here, we report five high resolution crystal structures of the catalytic core of Alkbh5 in complex with different ligands. Compared with other AlkB proteins, Alkbh5 displays several unique structural features on top of the conserved double-stranded β-helix fold typical of this protein family. Among the unique features, a distinct “lid” region of Alkbh5 plays a vital role in substrate recognition and catalysis. An unexpected disulfide bond between Cys-230 and Cys-267 is crucial for the selective binding of Alkbh5 to single-stranded RNA/DNA by bringing a “flipping” motif toward the central β-helix fold. We generated a substrate binding model of Alkbh5 based on a demethylation activity assay of several structure-guided site-directed mutants. Crystallographic and biochemical studies using various analogs of α-ketoglutarate revealed that the active site cavity of Alkbh5 is much smaller than that of FTO and preferentially binds small molecule inhibitors. Taken together, our findings provide a structural basis for understanding the substrate recognition specificity of Alkbh5 and offer a foundation for selective drug design against AlkB members.

[1]  Mingming Jia,et al.  COSMIC (the Catalogue of Somatic Mutations in Cancer): a resource to investigate acquired mutations in human cancer , 2009, Nucleic Acids Res..

[2]  Richard Bonneau,et al.  The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. , 2012, Molecular cell.

[3]  R J Roberts,et al.  Sequence specificity of the human mRNA N6-adenosine methylase in vitro. , 1990, Nucleic acids research.

[4]  Jef Rozenski,et al.  The RNA modification database, RNAMDB: 2011 update , 2010, Nucleic Acids Res..

[5]  P. Weber,et al.  Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB , 2006, Nature.

[6]  Hiroshi Yamamoto,et al.  Expression and sub-cellular localization of human ABH family molecules , 2007, Journal of cellular and molecular medicine.

[7]  T. Pan.  N6-methyl-adenosine modification in messenger and long non-coding RNA. , 2013, Trends in biochemical sciences.

[8]  C. Schofield,et al.  Structural studies on human 2-oxoglutarate dependent oxygenases. , 2010, Current opinion in structural biology.

[9]  Erling Seeberg,et al.  AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli , 2002, Nature.

[10]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[11]  O. Elemento,et al.  Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and near Stop Codons , 2012, Cell.

[12]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[13]  Tao Zhang,et al.  Direct-method SAD phasing of proteins enhanced by the use of intrinsic bimodal phase distributions in the subsequent phase-improvement process. , 2009, Acta crystallographica. Section D, Biological crystallography.

[14]  Chengqi Yi,et al.  N6-Methyladenosine in Nuclear RNA is a Major Substrate of the Obesity-Associated FTO , 2011, Nature chemical biology.

[15]  Chengqi Yi,et al.  Crystal structures of DNA/RNA repair enzymes AlkB and ABH2 bound to dsDNA , 2008, Nature.

[16]  Amy C Yan,et al.  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* , 2011, The Journal of Biological Chemistry.

[17]  J. Essigmann,et al.  Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[18]  A. Imbalzano,et al.  Protein Arginine Methyltransferase 7 Regulates Cellular Response to DNA Damage by Methylating Promoter Histones H2A and H4 of the Polymerase δ Catalytic Subunit Gene, POLD1* , 2012, The Journal of Biological Chemistry.

[19]  Airlie J. McCoy,et al.  Solving structures of protein complexes by molecular replacement with Phaser , 2006, Acta crystallographica. Section D, Biological crystallography.

[20]  P. Emsley,et al.  Features and development of Coot , 2010, Acta crystallographica. Section D, Biological crystallography.

[21]  Fei Long,et al.  BALBES: a molecular-replacement pipeline , 2007, Acta crystallographica. Section D, Biological crystallography.

[22]  Michael A McDonough,et al.  Role of the jelly-roll fold in substrate binding by 2-oxoglutarate oxygenases. , 2012, Current opinion in structural biology.

[23]  A. Brunger.  Version 1.2 of the Crystallography and NMR system , 2007, Nature Protocols.

[24]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[25]  J. Tainer,et al.  Human ABH3 structure and key residues for oxidative demethylation to reverse DNA/RNA damage , 2006, The EMBO journal.

[26]  Z. Otwinowski,et al.  Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[27]  Randy J. Read,et al.  Dauter Iterative model building , structure refinement and density modification with the PHENIX AutoBuild wizard , 2007 .

[28]  Arne Klungland,et al.  ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. , 2013, Molecular cell.

[29]  Chengqi Yi,et al.  Oxidative demethylation of 3‐methylthymine and 3‐methyluracil in single‐stranded DNA and RNA by mouse and human FTO , 2008, FEBS letters.

[30]  T. Hollis,et al.  Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching , 2010, PloS one.

[31]  N. Liabakk,et al.  Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA* , 2008, Journal of Biological Chemistry.

[32]  M. Mann,et al.  Lysine Acetylation Targets Protein Complexes and Co-Regulates Major Cellular Functions , 2009, Science.

[33]  G. Pfeifer,et al.  Repair of Methylation Damage in DNA and RNA by Mammalian AlkB Homologues* , 2005, Journal of Biological Chemistry.

[34]  Yun-Gui Yang,et al.  N6-methyl-adenosine (m6A) in RNA: An Old Modification with A Novel Epigenetic Function , 2012, Genom. Proteom. Bioinform..

[35]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[36]  C. Kahana,et al.  Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA. , 1979, Nucleic acids research.

[37]  Collaborative Computational,et al.  The CCP4 suite: programs for protein crystallography. , 1994, Acta crystallographica. Section D, Biological crystallography.

[38]  Robert P. Hausinger,et al.  Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage , 2002, Nature.

[39]  Chuan He,et al.  FTO-Mediated Formation of N6-Hydroxymethyladenosine and N6-Formyladenosine in Mammalian RNA , 2013, Nature Communications.

[40]  H. Krokan,et al.  AlkB demethylases flip out in different ways. , 2008, DNA repair.

[41]  B. Moss,et al.  N6, O2′-dimethyladenosine a novel methylated ribonucleoside next to the 5′ terminal of animal cell and virus mRNAs , 1975, Nature.

[42]  Maria Merino,et al.  Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome. , 2002, Cancer cell.

[43]  David C. Schwartz,et al.  DNA sequence of human chromosome 17 and analysis of rearrangement in the human lineage , 2006, Nature.

[44]  M. Kupiec,et al.  Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq , 2012, Nature.

[45]  Kevin Cowtan,et al.  The Buccaneer software for automated model building. 1. Tracing protein chains. , 2006, Acta crystallographica. Section D, Biological crystallography.

[46]  P. Ratcliffe,et al.  Human AlkB Homologue 5 Is a Nuclear 2-Oxoglutarate Dependent Oxygenase and a Direct Target of Hypoxia-Inducible Factor 1α (HIF-1α) , 2011, PloS one.

[47]  Michael A McDonough,et al.  Structural basis for inhibition of the fat mass and obesity associated protein (FTO). , 2013, Journal of medicinal chemistry.

[48]  Qiang Wang,et al.  Crystal structure of the FTO protein reveals basis for its substrate specificity , 2010, Nature.

[49]  Chris P. Ponting,et al.  The Obesity-Associated FTO Gene Encodes a 2-Oxoglutarate-Dependent Nucleic Acid Demethylase , 2007, Science.

[50]  Kevin Cowtan,et al.  The Buccaneer software for automated model building , 2006 .