Reading and erasing of the phosphonium analogue of trimethyllysine by epigenetic proteins

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.

Probing lysine posttranslational modifications by unnatural amino acids

“…6B, Phosphonium). 83 In another study, we demonstrated the need for L-stereochemistry for efficient lysine demethylation by KDMs. 67 However, KDMs were observed to be more accepting towards conformationally constrained lysine analogues, with demethylation being catalysed by KDM4 subfamily members for trans-C-4/C-5 dehydrolysine substrates (K E me2) (Fig.…”

“…5C, Phosphonium). 83 Increased binding affinities were observed for H3K P 4me3-reader complexes, indicating that it is possible that unnatural Kme3 mimics associate stronger with reader proteins.…”

Biochemical and Structural Insights into FIH‐Catalysed Hydroxylation of Transient Receptor Potential Ankyrin Repeat Domains

“…Peptide synthesis and purification : Peptides derived from TRPA1, TRPV3 and the 1CA consensus sequence [28] were synthesised with C‐terminal amides using a CEM Liberty Blue solid‐phase peptide synthesis (SPPS) machine using Oxyma®/DIC‐mediated, microwave‐assisted couplings as reported [43] . In brief, SPPS was performed using rink amide MBHA resin and 0.2 M solutions of FMOC‐protected amino acids in DMF.…”

Recognition of stapled histone H3K4me3 peptides by epigenetic reader proteins

“…11–13 Comparative binding analysis demonstrated a selectivity trend for higher methylation states (Kme3 > Kme2 > Kme > K) and for l -Kme3 over d -Kme3. 9,14 In addition, H3K4me3 readers have shown affinity towards cysteine-derived analogues of Kme3 in histone peptides and intact histone proteins, 15–18 and bind the simplest neutral carba analogue, 12 the positively charged phospha analogue, 19 and trimethyllysine analogues that differ in the length of the side chain. 20,21…”

“…[11][12][13] Comparative binding analysis demonstrated a selectivity trend for higher methylation states (Kme3 4 Kme2 4 Kme 4 K) and for L-Kme3 over D-Kme3. 9,14 In addition, H3K4me3 readers have shown affinity towards cysteine-derived analogues of Kme3 in histone peptides and intact histone proteins, [15][16][17][18] and bind the simplest neutral carba analogue, 12 the positively charged phospha analogue, 19 and trimethyllysine analogues that differ in the length of the side chain. 20,21 The backbone of the H3K4me3-containing histone tail assumes unstructured conformations in an unbound state, and is subjected to induced-fit conformational transitions to mediate intramolecular hydrogen-bonding, cation-p interactions and salt-bridges with the reader domain (Fig.…”