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

Protein Sci. 2010 Nov;19(11):2141-51. doi: 10.1002/pro.492.

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 Förster 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 multi-well 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Dimerization
  • Fluorescence Resonance Energy Transfer / methods*
  • Kinetics
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / metabolism
  • Models, Molecular
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / metabolism
  • Protein-Arginine N-Methyltransferases / chemistry*
  • Protein-Arginine N-Methyltransferases / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Repressor Proteins / chemistry*
  • Repressor Proteins / metabolism
  • S-Adenosylhomocysteine / chemistry
  • S-Adenosylhomocysteine / metabolism
  • S-Adenosylmethionine / chemistry*
  • S-Adenosylmethionine / metabolism

Substances

  • Luminescent Proteins
  • Nuclear Proteins
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • S-Adenosylmethionine
  • S-Adenosylhomocysteine
  • PRMT1 protein, human
  • PRMT6 protein, human
  • Protein-Arginine N-Methyltransferases