Oligomerization of protein arginine methyltransferase 1 and its effect on methyltransferase activity and substrate specificity.

Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.

Significant Loop Motions in the SsoPTP Protein Tyrosine Phosphatase Allow for Dual General Acid Functionality.

Conformational dynamics are important factors in the function of enzymes, including protein tyrosine phosphatases (PTPs). Crystal structures of PTPs first revealed the motion of a protein loop bearing a conserved catalytic aspartic acid, and subsequent nuclear magnetic resonance and computational analyses have shown the presence of motions, involved in catalysis and allostery, within and beyond the active site. The tyrosine phosphatase from the thermophilic and acidophilic Sulfolobus solfataricus (SsoPTP) displays motions of its acid loop together with dynamics of its phosphoryl-binding P-loop and the Q-loop, the first instance of such motions in a PTP. All three loops share the same exchange rate, implying their motions are coupled. Further evidence of conformational flexibility comes from mutagenesis, kinetics, and isotope effect data showing that E40 can function as an alternate general acid to protonate the leaving group when the conserved acid, D69, is mutated to asparagine. SsoPTP is not the first PTP to exhibit an alternate general acid (after VHZ and TkPTP), but E40 does not correspond to the sequence or structural location of the alternate general acids in those precedents. A high-resolution X-ray structure with the transition state analogue vanadate clarifies the role of the active site arginine R102, which varied in structures of substrates bound to a catalytically inactive mutant. The coordinated motions of all three functional loops in SsoPTP, together with the function of an alternate general acid, suggest that catalytically competent conformations are present in solution that have not yet been observed in crystal structures.