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J Biol Chem. 2015 Apr 10;290(15):9674-89. doi: 10.1074/jbc.M115.636894. Epub 2015 Feb 24.

Histone H2A and H4 N-terminal tails are positioned by the MEP50 WD repeat protein for efficient methylation by the PRMT5 arginine methyltransferase.

Author information

1
From the Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and.
2
JPT Peptide Technologies GmbH, Volmerstrasse 5, 12489 Berlin, Germany.
3
From the Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461 and david.shechter@einstein.yu.edu.

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.

KEYWORDS:

Enzyme Kinetics; Enzyme Mechanism; Histone Methylation; Peptide Array; Protein Arginine N-methyltransferase 5 (PRMT5); WD Repeat

PMID:
25713080
PMCID:
PMC4392268
DOI:
10.1074/jbc.M115.636894
[Indexed for MEDLINE]
Free PMC Article

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