Oligomerization of protein arginine methyltransferase 1 and its functional impact on substrate arginine methylation

Abstract

Protein arginine methyltransferases (PRMTs) are important posttranslational modifying enzymes in eukaryotic proteins and regulate diverse pathways from gene transcription, RNA splicing, and signal transduction to metabolism. Increasing evidence supports that PRMTs exhibit the capacity to form higher-order oligomeric structures, but the structural basis of PRMT oligomerization and its functional consequence are elusive. Herein, we revealed for the first time different oligomeric structural forms of the predominant arginine methyltransferase PRMT1 using cryo-EM, which included tetramer (dimer of dimers), hexamer (trimer of dimers), octamer (tetramer of dimers), decamer (pentamer of dimers), and also helical filaments. Through a host of biochemical assays, we showed that PRMT1 methyltransferase activity was substantially enhanced as a result of the high-ordered oligomerization. High-ordered oligomerization increased the catalytic turnover and the multimethylation processivity of PRMT1. Presence of a catalytically dead PRMT1 mutant also enhanced the activity of WT PRMT1, pointing out a noncatalytic role of oligomerization. Structural modeling demonstrates that oligomerization enhances substrate retention at the PRMT1 surface through electrostatic force. Our studies offered key insights into PRMT1 oligomerization and established that oligomerization constitutes a novel molecular mechanism that positively regulates the enzymatic activity of PRMTs in biology.

Keywords: PRMT1; arginine methylation; cryo-EM; enzyme activity regulation; molecular modeling; oligomerization; protein arginine methyltransferase; protein structure.

Figure 1

Structural characterization of PRMT1.A, surface view of 3D structures of various oligomerization states of PRMT1. B, surface view of the PRMT1 reconstruction superimposed with the subunit models; the subunits are represented in different colors. The filament has a right-handed helical symmetry with a rise of 23.34 Å and a 103.59° twist. C, the PRMT1 oligomer interface 2 region depicts the interface residues and interactions. Residues located at the interface 2 are shown and labeled. The interactions are shown as black lines. D, the PRMT1 oligomer interface 3 region depicts the interface residues. Residues located at the interface 3 are shown and labeled. The interactions are shown as black lines. Two distances are shown, indicating that the residues are not close enough to form strong interactions.

Figure 2

Oligomerization of PRMT1 detection by dynamic light scattering. Various concentrations of PRMT1 were incubated on ice for 10 min, then filtered by 0.2-micron syringe filter and collected data from the DLS. The top line with black circle indicates PRMT1 without denaturant. The open triangle and hexagon lines indicated PRMT1 oligomeric structure disrupted by 6 M guanidine hydrochloride and 5 M guanidine thiocyanate, respectively.

Figure 3

Determine the methylation state of R4 peptide by PRMT1 high-order oligomers. The reaction consisted of 0 μM (A), 0.5 μM (B), 1 μM (C) of PRMT1, 10 μM R4 peptide, and 50 μM SAM for 30 min at room temperature. The reaction was quenched with 5% TFA and subjected to MALDI analysis. D, the methylation percentage of individual peak in relative to R4 peptide.

Figure 4

Determine the methylation state of R4 peptide by PRMT1 and PRMT1-E153Q oligomers formation. PRMT1 and PRMT1-E153Q was incubated on ice for 20 min before the addition of 10 μM R4 and 50 μM of SAM. The reaction was at 25 °C on water bath for 30 min before quenching with 5% TFA. MALDI analysis shown the methylation events at different concentrations of PRMT1-E153Q. A, no PRMT1-WT or PRMT1-E153Q was added to the reaction. B, 0.5 μM of PRMT1-WT and no 0 PRMT1-E153Q to test for oligomerization without PRMT1-E153Q presence. C–E, contained 0.5 μM PRMT1-WT with 0.5 μM, 1 μM, and 2 μM PRMT1-E153Q, respectively. F, the methylation percentage of individual peak in relative to R4 peptide.

Figure 5

Determine the methylation state of H4 peptide by PRMT1 and PRMT1-E153Q oligomers formation. PRMT1 and PRMT1-E153Q was incubated on ice for 20 min before the addition of 10 μM H4 and 50 μM of SAM. The reaction was at 25 °C on water bath for 30 min before quenching with 5% TFA. MALDI analysis shown the methylation events at different concentrations of PRMT1-E153Q. A, 0.1 μM of PRMT1-WT and no PRMT1-E153Q to test for oligomerization without PRMT1-E153Q presence. B–D, contained 0.1 μM PRMT1-WT with 0.5 μM, 1 μM, and 2 μM PRMT1-E153Q, respectively. E, the methylation percentage of individual peak in relative to R4 peptide.

Figure 6

Arginine methylation of various peptides by high-order oligomeric PRMT1-WTmeasured using the radiometric filter-binding assay. Various concentrations of PRMT1-WT were incubated with radioactive 2 μM [3H]-SAM and 48 μM SAM and 50 μM of indicated peptides for 30 min. The reaction was quenched with 100% isopropanol. A–C, demonstrated PRMT1 methylation of peptide CPM. D–F, demonstrated CPM per monomer of PRMT1.

Figure 7

Arginine methylation of various peptides by high-order oligomeric PRMT1-WT and PRMT1 mutants. Filter-binding assay was used to characterize the methyltransferase activity. Firstly, 0.05 μM of PRMT1 was incubated with various concentrations of PRMT1-E153Q or PRMT1-H293G/E153Q on ice for 20 min. Afterward, 2 μM radioactive [3H]-SAM and 48 μM SAM and 50 μM of indicated peptides was added and incubated for 30 min before quenching the reaction with 100% isopropanol. A–C, showed PRMT1 methylation of peptides in CPM in the presence of PRMT1-E153Q. D–F, demonstrated CPM per monomer of PRMT1 in the presence of PRMT1-E153Q. G–I, showed PRMT1 methylation of peptide in CPM in the presence of PRMT1-H293G/E153Q. J–L, demonstrated CPM per monomer of PRMT1 in the presence of PRMT1-H293G/E153Q.

Figure 8

Steady state kinetic characterization of PRMT1 catalysis in oligomeric states. Radioactive kinetic parameters were measured using filter-binding assay. The reaction mixture consisted of 0.05 μM PRMT1-WT, 15 μM ¹⁴C-SAM, and various concentrations of Ac-H4(1–16) or Ac-H4(1–22) peptide. The reaction time was 15 min at 30 °C and quenched with 100% isopropanol. For the reaction with PRMT1-E153Q, 0.05 μM PRMT1-E153Q was incubated on ice with PRMT1 for 20 min equilibration before adding SAM and peptide. A, kinetic parameters of oligomeric states in graphical form. B, representation of the kinetic parameters in k_cat, K_0.5, and k_cat/K_0.5.

Figure 9

Oligomerization helps PRMT1 retain its substrate. The experiment was carried out by the SX20 stopped-flow spectrometers with (A) 0.5 μM, (B) 1.5 μM, and (C) 5 μM of PRMT1. The PRMT1 was premixed with 0.8 μM H4FL peptide. The mixture was diluted with the reaction buffer for a 1:25 dilution when the reaction was run. D, data summary of k_off.

Figure 10

Binding modes and kinetics of H4(21)-peptide association to PRMT1 oligomers. Zoomed-in view of the binding contacts formed by H4(21) peptide in its primary docking configuration with (A) monomer, (B) dimer, and (C) tetramer of PRMT1. The oligomers in the top panel are colored by domains as indicated in the inset of panel a. H4(21) peptide is depicted in light green. Docking conformations of the H4 peptide and surface electrostatics of the PRMT1 complexes for (D) monomer, (E) dimer, and (F) tetramer. The electrostatic potentials were computed with APBS and mapped onto the molecular surfaces in the range from -5k_bT/e (red) to +5k_bT/e (blue). G, a schematic representation of the BrownDye algorithm with decision boundaries being marked in red and dark green. H, Second-order rate constants k_on for H4 peptide binding to the different oligomers of PRMT1 as estimated from Brownian dynamics calculations.