Abstract
Nε-Methylation of lysine residues in histones plays an essential role in the regulation of eukaryotic transcription. The ‘highest’ methylation mark, Nε-trimethyllysine, is specifically recognised by Nε-trimethyllysine binding ‘reader’ domains, and undergoes demethylation, as catalysed by 2-oxoglutarate dependent JmjC oxygenases. We report studies on the recognition of the closest positively charged Nε-trimethyllysine analogue, i.e. its trimethylphosphonium derivative (KPme3), by Nε-trimethyllysine histone binding proteins and Nε-trimethyllysine demethylases. Calorimetric and computational studies with histone binding proteins reveal that H3KP4me3 binds more tightly than the natural H3K4me3 substrate, though the relative differences in binding affinity vary. Studies with JmjC demethylases show that some, but not all, of them can accept the phosphonium analogue of their natural substrates and that the methylation state selectivity can be changed by substitution of nitrogen for phosphorus. The combined results reveal that very subtle changes, e.g. substitution of nitrogen for phosphorus, can substantially affect interactions between ligand and reader domains / demethylases, knowledge that we hope will inspire the development of highly selective small molecules modulating their activity.
Highlights
3,6, Akane Kawamura[1,2], Nε-Methylation of lysine residues in histones plays an essential role in the regulation of eukaryotic transcription
Four identified non-catalytic domains interact with Nε-trimethyllysines: plant homeodomains (PHD) (Fig. 1c), tandem tudor domains (TTD), chromodomains (CHD), and malignant brain tumour (MBT) proteins[3], all of which bind Nε-trimethyllysine in a cage comprised of typically hydrophobic and aromatic residues[4]
The Fmoc-KPme3-OH and the Fmoc-Kme3-OH control were incorporated into human histone H3-tail fragment peptides (histone H3 residues 1-10, ART(KPme3)QTARKS: H3KP4me3/ ART(Kme3)QTARKS: H3K4me[3]; and histone H3 residues 1–15, ARTKQTAR(KPme3)STGGKA: H3KP9me3/ARTKQTAR(Kme3) STGGKA: H3K9me3) using Fmoc mediated solid-phase peptide synthesis (SPPS), followed by preparative HPLC (Supplementary Scheme 1)
Summary
3,6, Akane Kawamura[1,2], Nε-Methylation of lysine residues in histones plays an essential role in the regulation of eukaryotic transcription. The ‘highest’ methylation mark, Nε-trimethyllysine, is recognised by Nε-trimethyllysine binding ‘reader’ domains, and undergoes demethylation, as catalysed by 2-oxoglutarate dependent JmjC oxygenases. Studies with JmjC demethylases show that some, but not all, of them can accept the phosphonium analogue of their natural substrates and that the methylation state selectivity can be changed by substitution of nitrogen for phosphorus. Histone lysine-residues are amongst the most frequently modified of all residues, including by acylationtype modifications, most commonly acetylation[1] They are iteratively Nε-methylated to give Nε-monomethylated (Kme1), Nε-dimethylated (Kme2), and Nε-trimethylated lysine (Kme3) residues (Fig. 1a). Experimental and computational studies have shown that binding of Nεtrimethyllysine by readers is driven by cation–π interactions between the positively charged quaternary ammonium group of Nε-trimethyllysine and electron-rich aromatic residues and by release of water molecules from the cage[5–9]. DNA encoding for the PHD3 finger of the KDM5A demethylase can fuse with that of nuclear pore protein
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