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.
Highlights
PRMT5-MEP50 is an arginine methyltransferase with significant roles in development and cancer
Our previous work defined two distinct relative classes of MEP50 molecules within the PRMT5-MEP50 tetramer: the directly bound MEP50 and the cross-dimer MEP50 [22]. Both our solved X. laevis PRMT5-MEP50 structure and the solved human complex structure [23] demonstrated unique N- and C-terminal domains of PRMT5 connected by a loop
Based on its structural organization and residue conservation and our electron microscopy images of XlPRMT5-MEP50 complexed with substrate, we hypothesized that the cross-dimer MEP50 is responsible for organizing substrate for the PRMT5 catalytic domain
Summary
PRMT5-MEP50 is an arginine methyltransferase with significant roles in development and cancer. Mechanisms of PRMT5-MEP50 Histone Methylation accessible catalytic site These observations are consistent with a conserved and significant stringency in substrate selection. We employ structural analysis as well as qualitative and quantitative methylation assays to measure enzymatic activity and binding affinity for histones to PRMT5-MEP50. Our results support the concept that MEP50 interacts directly with histones and the N-terminal domain of PRMT5 but may contribute to active site remodeling in the C-terminal domain of PRMT5 for efficient methyl transfer. Our computational modeling revealed multiple modes of substrate-enzyme interaction consistent with our experimental data These studies support the essential function of MEP50 in binding histone fold and presenting histone tail substrate to the active site of PRMT5 cross-dimer for efficient methylation
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