Installation of Trimethyllysine Analogs on Intact Histones via Cysteine Alkylation

Abstract

Site-specific incorporation of post-translationally modified amino acids into proteins, including histones, has been a subject of great interest for chemical and biochemical communities. Here, we describe a site-specific incorporation of structurally simplest trimethyllysine analogs into position 4 of the intact histone H3 protein. An efficient alkylation of cysteine 4 of the recombinantly expressed histone H3 provides a panel of trimethyllysine analogs that differ in charge, charge density, sterics, and chain length. We demonstrate that H3 histone that bears trimethyllysine analogs can be further assembled into the octameric histone complex that constitutes the nucleosome. Binding studies showed that H3 histone that possesses trimethyllysine analogs is well recognized by a PHD3 reader domain of human JARID1A. This work provides important (bio)­chemical tools for fundamental biomolecular studies aimed at unravelling the molecular basis of the higher order nucleosome and chromatin assemblies.

γ-Thialysine versus Lysine: An Insight into the Epigenetic Methylation of Histones

“…At present, however, it is not clear how well do unnatural cysteine-derived amino acids resemble natural unmodified and posttranslationally modified amino acids. 20,23,24,26 There are some conflicting quantitative data on whether epigenetic reader proteins recognize the cysteine-derived γ-trimethylthialysine (KCme3) and trimethyllysine (Kme3) equally well, [27][28][29] while KCme3 appears to be a poor substrate for histone lysine demethylases. 30 Enzymatic assays using [ 3 H-Me]-SAM showed that histones bearing γ-thialysine (KC), the cysteine-derived γ-thialysine analog, underwent the SUV39H1 methyltransferase-catalyzed methylation reaction.…”

Mechanism of biomolecular recognition of trimethyllysine by the fluorinated aromatic cage of KDM5A PHD3 finger

“…Despite different folding patterns, these reader proteins have a common feature as they all possess electron-rich aromatic cages, most often comprised of 1-4 side chains of Phe, Tyr and Trp, although some cages also include the negatively charged Asp or Glu residues 13,14 . Comparative binding studies between trimethyllysine and its neutral carba analogue led to the conclusion that the recognition of the positively charged trimethyllysine by the aromatic cage containing readers is predominantly driven by a combination of favourable cation-π interactions and the release of high-energy water molecules located inside the aromatic cages [15][16][17] .…”

Advances and Opportunities in Epigenetic Chemical Biology

“…Cysteine residues contain a reactive thiol group, rendering them very amenable to conjugation chemistries (Figure 16, top). Fortunately, histones H2A, H2B, and H4 do not contain any native Cys residues, and H3 contains just one or two Cys residues, depending on isoform, that can be mutated to Ala or Ser with no functional consequences [387] . Together, this allows any histone residue to be mutated to a Cys for site‐specific labelling.…”

“…Cysteine conjugations have proved useful for installing PTMs and PTM analogues, particularly those of lysine in histones [388] . In recent years, a wide variety of trimethyllysine analogues have been installed in H3 through Cys alkylation [387] . Hydrazide analogues of acetyl‐, hydroxyisobutyryl‐, and ubiquityllysine have also been installed at sites 56 and 122 in H3 by Cys mutation, showing that hydroxyisobutyrylated and ubiquitinylated K56 increase DNA unwrapping, and at K122, all three PTMs destabilize the nucleosome [389] .…”