Forster resonance energy transfer measurements of cofactor-dependent effects on protein arginine N-methyltransferase homodimerization

Protein arginine N-methyltransferase (PRMT) dimerization is required for methyl group transfer from the cofactor S-adenosyl-L-methionine (Ado Met) to arginine residues in protein substrates, forming S-adenosyl-L-homocysteine (AdoHcy) and methylarginine residues. In this study, we use Forster resonance energy transfer (FRET) to determine dissociation constant (KD) values for dimerization of PRMT1 and PRMT6. By attaching monomeric Cerulean and Citrine fluorescent proteins to their N-termini, fluorescent PRMTs are formed that exhibit similar enzyme kinetics to unconjugated PRMTs. These fluorescent proteins are used in FRET-based binding studies in a multi-well format. In the presence of Ado Met, fluorescent PRMT1 and PRMT6 exhibit 4- and 6-fold lower dimerization KD values, respectively, than in the presence of AdoHcy, suggesting that Ado Met promotes PRMT homodimerization in contrast to AdoHcy. We also find that the dimerization KD values for PRMT1 in the presence of Ado Met or AdoHcy are, respectively, 6- and 10-fold lower than the corresponding values for PRMT6. Considering that the affinity of PRMT6 for AdoHcy is 10-fold higher than for Ado Met, PRMT6 function may be subject to cofactor-dependent regulation in cells where the methylation potential (i.e., ratio of Ado Met to AdoHcy) is low. Since PRMT1 affinity for Ado Met and AdoHcy is similar, however, a low methylation potential may not affect PRMT1 function.