Förster resonance energy transfer measurements of cofactor‐dependent effects on protein arginine N‐methyltransferase homodimerization

Abstract

Protein arginine N-methyltransferase (PRMT) dimerization is required for methyl group transfer from the cofactor S-adenosyl-L-methionine (AdoMet) to arginine residues in protein substrates, forming S-adenosyl-L-homocysteine (AdoHcy) and methylarginine residues. In this study, we use Fö rster resonance energy transfer (FRET) to determine dissociation constant (K D ) 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 multiwell format. In the presence of AdoMet, fluorescent PRMT1 and PRMT6 exhibit 4-and 6-fold lower dimerization K D values, respectively, than in the presence of AdoHcy, suggesting that AdoMet promotes PRMT homodimerization in contrast to AdoHcy. We also find that the dimerization K D values for PRMT1 in the presence of AdoMet 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 AdoMet, PRMT6 function may be subject to cofactor-dependent regulation in cells where the methylation potential (i.e., ratio of AdoMet to AdoHcy) is low. Since PRMT1 affinity for AdoMet and AdoHcy is similar, however, a low methylation potential may not affect PRMT1 function.

Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

“…Our observations that both cofactor and enzyme concentration affect PRMT1 processivity led us to question what influences the degree to which PRMT1 acts processively. From a mechanistic point of view, we know that higher concentrations of PRMT1 revealed a correspondingly increased level of dimerization and higher order oligomers . Therefore, we hypothesized that factors that enhance enzyme dimerization might in turn increase processivity.…”

“…We have previously argued that PRMT1 in solution contains concentration‐dependent enzyme populations in various oligomeric states whose kinetic behaviors differ . At a lower enzyme concentration, PRMT1 was susceptible to substrate inhibition, whereas at a high concentration—well above the K D value of dimerization (110 n m )—a kinetically distinct PRMT1 population was no longer susceptible to substrate inhibition. As the inhibition effect on H4‐OH peptide is alleviated at higher enzyme concentrations, our results further support our prevailing theory.…”

“…CARM1 phosphorylation at a conserved serine residue near the dimer interface was shown to inhibit dimerization and prevent SAM binding, thereby regulating enzyme function during cell cycle progression . In addition to dimerization being a requirement for SAM binding, our group found that SAM and SAH influenced the dissociation constants for PRMT1 and PRMT6 homodimers . PRMT dimerization and its relationship with cofactor binding, therefore, are undoubtedly important aspects of enzyme activity.…”

Automethylation of protein arginine methyltransferase 6 (PRMT6) regulates its stability and its anti-HIV-1 activity

“…PRMT6 requires homodimerization to transfer a methyl group from SAM to the protein substrate and this could favour automethylation [27]. As documented for CARM1 (PRMT4), another member of the PRMT family, we have hypothesized that automethylation could modulate PRMT6 function [28].…”

Protein Arginine N‐Methyltransferase Substrate Preferences for Different Nη‐Substituted Arginyl Peptides

“…[15] 14 C Methylation and tricine gel separation of the R1 peptide series: Enzymes were prepared as previously described. [15,22,25,36] The PRMT1 D51N variant was created from pET-28(+)-PRMT1 using QuikChange (Agilent Technologies) and primers D51N-F (5'-CCA CGA GGA GAT GCT AAA GAA TGA G-3') and D51N-R (5'-CGT GAG GGT TCG CAC CTC ATT CTT TA-3'). R1 peptide (250 mm) was incubated in methylation buffer (HEPES (50 mm pH 8.0), NaCl (10 mm), dithiothreitol (DTT, 1 mm)) [25] with S-adenosyl-l-[methyl- ), and PRMT1, PRMT4, PRMT6, PRMT1 E153Q, or PRMT1 D51N (2 mm) for 16 h at 37 8C.…”