Probing multiple enzymatic methylation events in real time with NMR spectroscopy

Abstract

Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated ¹³C into the donor methyl group of the enzyme cofactor S-adenosyl methionine. In this way, we may study enzymatic methylation by monitoring the appearance of distinct resonances corresponding to mono-, di-, or trimethyl lysine without the need to isotopically enrich the substrate. To demonstrate the capabilities of this method, we evaluated the activity of three lysine methyltransferases, Set7, MWRAD₂ (MLL1 complex), and PRDM9, toward the histone H3 tail. We monitored mono- or multimethylation of histone H3 tail at lysine 4 through sequential short two-dimensional heteronuclear single quantum coherence experiments and fit the resulting progress curves to first-order kinetic models. In summary, NMR detection of PTMs in one-pot, real-time reaction using facile cofactor isotopic enrichment shows promise as a method toward understanding the intricate mechanisms of methyltransferases and other enzymes.

Figure 1

¹³C-enriched SAM allows detection of methyl-lysine over time. (A) Structure of S-adenosyl methionine (SAM) depicting ¹³C enrichment on the donor methyl group. (B) Scheme describing lysine methylation by Set7 (¹³C-methyl group shown in red). (C) Endpoint matrix-assisted desorption/ionization-time-of-flight mass spectrum of unmodified H3 (2183 m/z, black) and ¹³C-methylated H3 (2197 m/z). (D) Endpoint NMR spectrum of ¹³C-monomethylated H3. The peak corresponding to unreacted SAM (black) is distinct from the peak corresponding to ¹³C-monomethylated H3 (red). (E) Sample time points of HSQC spectra showing buildup of monomethyl peak (red) and modest depletion of SAM peak (black). To see this figure in color, go online.

Figure 2

Substrate methylation using ¹³C-SAM yields distinct peaks corresponding to mono-, di-, and tri-methyl-lysine. (A) Color-coded methylation scheme by a tri-methyltransferase enzyme. (B) 2D ¹H, ¹³C-HSQC spectra showing methyl-lysine resonances at final experiment time points (red = Set7, blue = MLL1, and orange = PRMD9). (C) Sample time points of HSQC spectra showing the resonance intensities of the mono- and dimethyl peaks over time installed by MLL1. (D) Sample time points of PRDM9-installed mono-, di-, and trimethyl marks. To see this figure in color, go online.

Figure 3

Kinetic profiles and fits for mono-, di-, and trimethylation. (A) Progress curve (red triangles) and nonlinear fit (solid line) to the signal from Set7-monomethylated H3 peptide over time. (B) Progress curve tracking H3 mono- (red triangles) and di-methylation (blue circles) by MLL1. Nonlinear fits are shown by the solid lines. (C) Progress curves for mono- (red triangles), di- (blue circles), and trimethylation (orange squares) activity of PRDM9 toward H3K4. Fits are shown as solid lines. Rate constants extracted from global fits of methyltransferase data are shown in Table 1. Residuals for each fit are plotted below each progress curve panel. To see this figure in color, go online.

Figure 4

Multimethyltransferase activity toward H3K4Me1. (A) Representative progress curve tracking the disappearance of H3K4Me1 and appearance of H3K4Me2 catalyzed by MLL1. Mono- and dimethyl data points are red triangles and blue circles, respectively, and their nonlinear fits are shown as solid lines. The data were globally fit to a three-state model to extract a dimethyl rate constant, k₂. (B) Representative plot tracking trimethylation of H3K4Me1 by PRMD9. Monomethyl data and fit are shown in red, dimethyl data and fit are blue, and trimethyl data and fit are orange. The data were globally fit to a four-state model to extract di- and trimethyl rate constants (k₂ and k₃, respectively). Residuals for each fit are plotted by color beneath each panel. Rate constants from global fitting of each data set are shown in Table 1. To see this figure in color, go online.