title: Histone H2A and H4 N-terminal tails are positioned by the MEP50 WD repeat protein for efficient methylation by the PRMT5 arginine methyltransferase.
abstract: The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1-20) and H4(1-20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate Km, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.
authors.affiliation: From the Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and.
authors.forename: Emmanuel S
authors.initials: ES
authors.lastname: Burgos
authors.affiliation: From the Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and.
authors.forename: Carola
authors.initials: C
authors.lastname: Wilczek
authors.affiliation: From the Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and.
authors.forename: Takashi
authors.initials: T
authors.lastname: Onikubo
authors.affiliation: From the Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Bonanno
authors.affiliation: JPT Peptide Technologies GmbH, Volmerstrasse 5, 12489 Berlin, Germany.
authors.forename: Janina
authors.initials: J
authors.lastname: Jansong
authors.affiliation: JPT Peptide Technologies GmbH, Volmerstrasse 5, 12489 Berlin, Germany.
authors.forename: Ulf
authors.initials: U
authors.lastname: Reimer
authors.affiliation: From the Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and david.shechter@einstein.yu.edu.
authors.forename: David
authors.initials: D
authors.lastname: Shechter
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011484:Protein-Arginine N-Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D029742:Caenorhabditis elegans Proteins; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D011484:Protein-Arginine N-Methyltransferases; D029867:Xenopus Proteins; C570111:methylosome protein 50, Xenopus; C400529:PRMT5 protein, human; C495466:Prmt5 protein, Xenopus; C478359:WDR77 protein, human; D014157:Transcription Factors
keywords: Enzyme Kinetics; Enzyme Mechanism; Histone Methylation; Peptide Array; Protein Arginine N-methyltransferase 5 (PRMT5); WD Repeat