Fine-tuning of lysine side chain modulates the activity of histone lysine methyltransferases

Abstract

Histone lysine methyltransferases (KMTs) play an important role in epigenetic gene regulation and have emerged as promising targets for drug discovery. However, the scope and limitation of KMT catalysis on substrates possessing substituted lysine side chains remain insufficiently explored. Here, we identify new unnatural lysine analogues as substrates for human methyltransferases SETD7, SETD8, G9a and GLP. Two synthetic amino acids that possess a subtle modification on the lysine side chain, namely oxygen at the γ position (K_O, oxalysine) and nitrogen at the γ position (K_N, azalysine) were incorporated into histone peptides and tested as KMTs substrates. Our results demonstrate that these lysine analogues are mono-, di-, and trimethylated to a different extent by trimethyltransferases G9a and GLP. In contrast to monomethyltransferase SETD7, SETD8 exhibits high specificity for both lysine analogues. These findings are important to understand the substrate scope of KMTs and to develop new chemical probes for biomedical applications.

Figure 1

Histone lysine methylation. (a) Histone lysine methyltransferase (KMT)-catalyzed methylation of lysine in the presence of SAM cosubstrate. (b) View from a crystal structure of GLP complexed with the H3K9me2 histone peptide and SAH (PDB ID: 2RFI). (c) Lysine analogues K_N and K_O examined in the current study.

Figure 2

Synthesis of Fmoc-K_O(Boc)-OH (1).

Figure 3

Synthesis of Fmoc-Kn(Boc)₂-OH (2).

Figure 4

Representative structures of histone H3K9, H3K4, and H4K20 peptides where the natural lysine has been replaced by both γ-oxalysine and γ-azalysine at specific positions for KMT catalysis.

Figure 5

KMT-catalyzed methylation of lysine and its analogues that possess O and NH functionalities at the γ-position. MALDI-TOF MS data showing methylation of histone peptides in the presence of GLP with (a) H3K9, (b) H3K_O9, (c) H3K_N9; and G9a with (d) H3K9, (e) H3K_O9, (f) H3K_N9; and SETD8 with (g) H4K20, (h) H4K_O20, (i) H4K_N20; and SETD7 with (j) H3K4, (k) H3K_O4, (l) H3K_N4. Red spectra show 1 h reactions at 37 °C including KMTs (2 µM), histone peptide (100 µM) and SAM (500 µM for GLP/G9a and 200 µM for SETD8/SED7) and black spectra the no-enzyme controls.

Figure 6

NMR analyses of GLP-catalyzed methylation reactions. ¹H NMR of (a) H3K9; (b) H3K_O9; and (c) H3K_N9 spectra revealing methylation of histone peptides in the presence of GLP and SAM. ¹H-¹³C HSQC data of (d) H3K9; (e) H3K_O9; and (f) H3K_N9 data with the assignment of cross-peaks. Correlations corresponding to the methylated lysine analogues are highlighted.