The nucleophilic amino group of lysine is central for histone lysine methyltransferase catalysis

Abstract

Histone lysine methyltransferases (KMTs) are biomedically important epigenetic enzymes that catalyze the transfer of methyl group from S-adenosylmethionine to lysine’s nucleophilic ε-amino group in histone tails and core histones. Understanding the chemical basis of KMT catalysis is important for discerning its complex biology in disease, structure-function relationship, and for designing specific inhibitors with therapeutic potential. Here we examine histone peptides, which possess simplest lysine analogs with different nucleophilic character, as substrates for human KMTs. Combined MALDI-TOF MS experiments, NMR analyses and molecular dynamics and free-energy simulations based on quantum mechanics/molecular mechanics (QM/MM) potential provide experimental and theoretical evidence that KMTs do have an ability to catalyze methylation of primary amine-containing N-nucleophiles, but do not methylate related amide/guanidine-containing N-nucleophiles as well as simple O- and C-nucleophiles. The results demonstrate a broader, but still limited, substrate scope for KMT catalysis, and contribute to rational design of selective epigenetic inhibitors.

Methylation of geometrically constrained lysine analogues by histone lysine methyltransferases

“…However, the other singlet observed at 2.64 ppm was directly coupled to a 13 C resonance at 42.6 ppm. The upfield shift in both the 1 H and 13 C resonances (B0.5 ppm for 1 H and B10 ppm for 13 C as compared to the other analogues) is consistent with dimethylation of H3K Z 9 and literature reports of H3K9me2 and H3hK9me2. Results from GLP-catalysed methylation on H3K Z 9 in the NMR assay were consistent with what was observed by MALDI-TOF MS data in the presence of increased amounts of GLP (Fig.…”

“…The downfield chemical shift of the 13 C methyl groups conclusively identified these new resonances as part of an NMe 3 + moiety, indicating that the lysine analogue was trimethylated, and matched the result obtained for H3K9. Further NMR spectroscopic analysis using 1 H-13 C HMBC and 1 H-1 H ROESY experiments confirmed the methylation of H3K E 9 and H3K yne 9 occurred at the 9 position due to the long-range 1 H- 13 C coupling between the trimethylammonium group and the C e of the histone peptide, as well as long-range through-space interactions between the 1 Hs of the trimethylammonium and the 1 Hs of e-CH 2 (Fig. S33-S39, ESI †).…”

“…S32b and d, ESI †). Subsequent edited 1 H- 13 C HSQC experiments revealed these signals as methyl groups with corresponding carbon resonances at 52.3 and 52.5 ppm, respectively ( Fig. 3b and d).…”

“…6,8 Recent studies have expanded the knowledge concerning the biocatalytic activity of human KMTs via examining various lysine analogues as substrates, revealing that KMTs not only display a high degree of specificity towards lysine, but also to simple lysine analogues. [9][10][11][12][13][14][15][16] Despite the progress in understanding of the role of the substrate-binding pocket on KMT catalysis, it is presently unclear how does the flexible lysine side chain contributes to substrate efficiency. In the ternary complex, the histone lysine side chain typically exhibits the anti (zig-zag) conformation, presumably enabling an optimal positioning towards the SAM cosubstrate for efficient methyl transfer (Fig.…”

“…S15a, ESI †), in line with recent studies. [9][10][11][12][13] Under the same conditions, the other six histone peptides containing the unsaturated K E , K Z , and K yne lysine analogues were examined.…”