Examining sterically demanding lysine analogs for histone lysine methyltransferase catalysis

Abstract

Methylation of lysine residues in histone proteins is catalyzed by S-adenosylmethionine (SAM)-dependent histone lysine methyltransferases (KMTs), a genuinely important class of epigenetic enzymes of biomedical interest. Here we report synthetic, mass spectrometric, NMR spectroscopic and quantum mechanical/molecular mechanical (QM/MM) molecular dynamics studies on KMT-catalyzed methylation of histone peptides that contain lysine and its sterically demanding analogs. Our synergistic experimental and computational work demonstrates that human KMTs have a capacity to catalyze methylation of slightly bulkier lysine analogs, but lack the activity for analogs that possess larger aromatic side chains. Overall, this study provides an important chemical insight into molecular requirements that contribute to efficient KMT catalysis and expands the substrate scope of KMT-catalyzed methylation reactions.

Figure 1

(a) Methylation of lysine residues by histone lysine methyltransferase in the presence of SAM cosubstrate. (b) View on the crystal structures of SETD8 complexed with H4K20 (green) and SAH (yellow) (left), and GLP complexed with H3K9me (green) peptide and SAH (yellow) (right). (c) A panel of sterically demanding lysine analogs.

Figure 2

Synthetic strategy for preparation of Fmoc-K_CP(Boc)-OH 1.

Figure 3

MALDI-TOF MS data showing SAM-dependent methylation of histone peptides in the presence of (a) SETD8 and (b) GLP. H4K20/H3K9 (first panels), H4K_CP20/H3K_CP9 (second panels), H4K_ba20/H3K_ba9 (third panels), H4F_3a20/H3F_3a9 (fourth panels), H4F_4a20/H3F_4a9 (fifth panels), H4A_P20/H3A_P9 (sixth panels), and H4Y20/H3Y9 (seventh panels). (Black = control reaction showing the histone peptide in the absence of KMT, red = KMT-catalyzed reaction).

Figure 4

Inhibition of SETD7, G9a and GLP (100 nM) in the presence of 100 µM of H3K*4 (SETD7) or H3K*9 (G9a/GLP) peptides.

Figure 5

¹H NMR spectra showing methylations of histone peptides (400 µM) in the presence GLP (8 µM) and SAM (2 mM). (a) H3K9; (b) H3K_CP9; (c) H3K_ba9; (d) H3F_3a9; (e) H3F_4a9; (f) H3A_P9; (g) H3Y9; (h) ¹H-¹³C HSQC data of H3K9 with the assignment of cross-peaks; (i) ¹H-¹³C HSQC data of H3K_CP9 with the assignment of cross-peaks.

Figure 6

(a) Free energy (potential of mean force) profiles for the first methylation reaction in SETD8 involving K, two K_CP and F_3a as a function of the reaction coordinate [R = r(C_M···S_δ) – r(C_M···N_ζ)]. Blue: H4K20 with a free energy barrier of 19.4 kcal mol⁻¹; Orange: K_CP with a barrier of 20.0 kcal mol⁻¹; Gray: another K_CP with a barrier of 19.3 kcal mol⁻¹; Yellow: F_3a with a barrier of 25.1 kcal mol⁻¹. (b) Representative active site structure of the reactant complex of SETD8 containing one of the two K_CP corresponding the orange line in Fig. 6a (see also the chemical structure inserted). The distribution map on the right shows the alignment of N_ζH₂ and the transferable methyl group in the reactant complex in terms of the distance (r) between N_ζ and C_M and the angle (θ) between the direction of electron lone pair on N_ζ and the C_M-S bond. (c) Representative active site structure of the reactant complexes of SETD8 with F_3a. (d) Representative active site structure of the near transition state for the methylation involving F_3a.

Figure 7

(a) Free energy (potential of mean force) profiles for the first, second and third methylation reactions in GLP involving one of the two K_CP molecules (see the structure inserted) as a function of the reaction coordinate [R = r(C_M···S_δ) – r(C_M···N_ζ)]; the results for the other K_CP are giving in the Supporitng Information. Blue: the first methyl transfer with a free energy barrier of 18.3 kcal mol⁻¹; Orange: the second methyl transfer with a barrier of 18.5 kcal mol⁻¹; Gray: the third methyl transfer with a barrier of 18.4 kcal mol⁻¹. (b) Representative active site structure of the reactant complex of GLP containing K_CP for the first methyl transfer. (c) Representative active site structure of the reactant complex of GLP for the third methyl transfer. (d) Representative active site structure of the near transition state for the third methyl transfer.