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

Abstract

N ^ε-Methylation of lysine residues in histones plays an essential role in the regulation of eukaryotic transcription. The 'highest' methylation mark, N ^ε-trimethyllysine, is specifically recognised by N ^ε-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 N ^ε-trimethyllysine analogue, i.e. its trimethylphosphonium derivative (K_Pme₃), by N ^ε-trimethyllysine histone binding proteins and N^ε-trimethyllysine demethylases. Calorimetric and computational studies with histone binding proteins reveal that H3K_P4me₃ 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.

Fig. 1. Demethylation and recognition of N^ε-methylated lysines by erasing enzymes and reader proteins.

a JmjC KDMs catalyse demethylation of N^ε-trimethyllysine residues. Our work explored recognition of the simplest positively charged N^ε-trimethyllysine analogue, i.e., the trimethylphosphonium derivative, by N^ε-methyllysine binding proteins and N^ε-methyllysine demethylases. n: number of methyl groups (3–1). b View from a structure of a JmjC KDM (KDM4A_JmjC, light blue) complexed with H3K9me₃ (yellow) and NOG (N-oxalylglycine, a 2OG analogue, white) (PDB: 2OQ6). c View from a structure of a reader (TAF3_PHD, purple) complexed with H3K4me₃ (yellow) (PDB: 2K17). Nitrogen: dark blue; oxygen: red; sulphur: yellow; zinc: grey; nickel: orange.

Fig. 2. Thermodynamic analyses of binding.

Representative ITC results from the interaction of (a) KDM5A_PHD3 and (b) KDM4A_TTD with H3K4me₃ (black) or H3K_P4me₃ (red) substrates. Top panels show the raw ITC data and the bottom panels show the processed results.

Fig. 3. MD simulation studies for reader SGF29_TTD.

Snapshots of simulations for SGF29_TTD complexed with a histone H3 fragment (liquorice) containing K_P4me₃ (cyan) or K4me₃ (white) at 0 ns and 10 ns.

Fig. 4. Analysis of Kme₃ and K_Pme₃ interactions with TRP2, a model for the KDM5A_PHD3 reader.

The TRP2 model employs the two tryptophan residues found in KDM5A_PHD3 (W1625, W1635). a Calculated VDD atomic charges (in mili-a.u.) for H3K4me₃ and H3K_P4me₃ (red: negative, blue: positive). b Frontier orbitals (with orbital energies in eV) for Kme₃, K_Pme₃, and TRP2, (isosurface drawn at 0.03), computed at the BLYP-D3BJ/TZ2P level using an X-ray structure for TRP2 (PDB: 3GL6).

Fig. 5. KDM4E_JmjC catalyses demethylation of H3K_P9me₃ to give H3K_P9me₂.

a KDM4E_JmjC catalysed demethylation of H3K_P9me₃. b Mass spectra and time-course analysis of H3K9me₃ (6.0 µM) and KDM4E_JmjC at 0 min (black) and 12 min (red) showing the substrate H3K9me₃ (orange) and demethylated products H3K9me₂ (purple) and H3K9me₁ (green). Mass spectra and time-course analysis of H3K_P9me₃ (5.0 µM) and KDM4E_JmjC (c 0.5 µM, d 5.0 µM) at time points 0 (red), 5 (green) and 60 min (red) acquired using MALDI-TOF MS. Conditions: Asc (500 µM), Fe(II) (50 µM) and 2OG (100 µM). Errors represent standard deviations (n = 2 or 3).

Fig. 6. KDM4E_JmjC catalyses demethylation of H3K_P9me₃ to give H3K_P9me₂H and H3K_P9me₂O as monitored by ³¹P NMR and ¹H-³¹P HMBC.

a ³¹P NMR analyses of H3K_P9me₃ (◊) incubated with KDM4E, addition of acid (top) or quenched by heating (bottom). Evidence for formation of H3K_P9me₂H (□, δ_P = 28.4 ppm, top) and H3K_P9me₂O (○, δ_P = 55.6 ppm, bottom). b Overlay of ¹H-³¹P HMBC analyses of the solutions after of H3K_P9me₃ incubated with KDM4E_JmjC, quenched by heating (green cross peaks), or addition of acid (red cross peaks). Experimental conditions: H3K_P9me₃ (500 µM), Asc (1.00 mM), 2OG (1.00 mM), Fe(II) (100 µM), KDM4E_JmjC (50 µM).