title: Substrate specificity of human protein arginine methyltransferase 7 (PRMT7): the importance of acidic residues in the double E loop.
abstract: Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-N(G)-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease.
authors.affiliation: From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095-1569.
authors.forename: You
authors.initials: Y
authors.lastname: Feng
authors.affiliation: From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095-1569.
authors.forename: Andrea
authors.initials: A
authors.lastname: Hadjikyriacou
authors.affiliation: From the Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095-1569 clarke@mbi.ucla.edu.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Clarke
mesh_terms: D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics; D002412:Cations; D002852:Chromatography, Ion Exchange / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D018698:Glutamic Acid / Q000235:genetics* / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011484:Protein-Arginine N-Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D002412:Cations; D006657:Histones; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D018698:Glutamic Acid; C487994:PRMT7 protein, human; D011484:Protein-Arginine N-Methyltransferases; D005982:Glutathione Transferase
keywords: Histone; Monomethylarginine; PRMT; Post-translational Modification (PTM); Protein Arginine Methyltransferase; Protein Arginine N-Methyltransferase 5 (PRMT5); Protein Methylation; S-Adenosylmethionine (AdoMet)