Investigation of the β-Sheet Interactions between dHP1 Chromodomain and Histone 3

Abstract

Methylated lysine 9 on the histone 3 (H3) tail recruits heterochromatin protein 1 from Drosophila (dHP1) via its chromodomain and results in gene silencing. The dHP1 chromodomain binds H3 K9Me3 with an aromatic cage surrounding the trimethyllysine. The sequence selectivity of binding comes from insertion of the histone tail between two β-strands of the chromodomain to form a three-stranded β-sheet. Herein, we investigated the sequence selectivity provided by the β-sheet interactions and how those interactions compare to other model systems. Residue Thr6 of the histone tail forms cross-strand interactions with Ala25 and Asp62 of the chromodomain. Each of these three residues was substituted for amino acids known to have high β-sheet propensities and/or to form favorable side chain-side chain (SC-SC) interactions in β-sheets, including hydrophobic, H-bonding, and aromatic interactions. We found that about 50% of the chromodomain mutants resulted in equal or tighter binding to the histone tail and about 25% of the histone tail mutants provided tighter binding compared to that of the native histone tail sequence. These studies provide novel insights into the sequence selectivity of the dHP1 chromodomain for the histone tail and relates the information gleaned from model systems and statistical studies to β-sheet-mediated protein-protein interactions. Moreover, this work suggests that the development of designer histone-chromodomain pairs for chemical biology applications is feasible.

SETD7 Regulates the Differentiation of Human Embryonic Stem Cells

“…Conformational changes associated with posttranslational modifications and characterized by an increase in β-structure at the C-terminal domain were previously described for H1 bound to DNA and in chromatin [ 54 , 55 ]. Recognition of methylated positions in H3 by several chromatin readers, including HP1, are mediated by β-sheet protein−protein interactions [ 56 , 57 ], so conformational changes induced by H1 methylation could affect interactions with consequences on chromatin organization or compaction. Alternatively, H1 methylation by SETD7 may influence its turnover or exchange.…”

Engineered Reader Proteins for Enhanced Detection of Methylated Lysine on Histones

“…To evaluate the possibility of developing an engineered reader protein as a detection reagent, we initiated our studies with the HP1α chromodomain from D. melanogaster as there have been significant mutagenesis studies probing its binding mechanism and selectivity. − The HP1α chromodomain recognizes H3K9me3 with a binding affinity of 14 μM. The molecular determinants of binding consist of sequestration of the Kme3 side chain in an aromatic cage as well as recognition of the surrounding sequence within a 3-stranded β-sheet. , Specific cross strand interactions within the β-sheet region impart selectivity for the histone sequence favoring H3K9me3 over neighboring H3K27me3, which differ by a single amino acid at the i – 3 position relative to Kme3.…”

“…1a). 38 Additionally, mutation of two residues near the aromatic cage, K46A and E52D, also improved binding by 10-fold and 2-fold, respectively, due to optimized electrostatic interactions (Fig. 1b).…”

“…1b). 37 The binding affinity of each of these single mutations had previously been measured by fluorescence anisotropy 38 , so we began with confirming their effects on binding using isothermal titration calorimetry (ITC, Fig. 1c).…”

“…We previously identified single point mutations in proximity to the aromatic cage and the β-sheet region that result in improved binding affinity to H3K9me3, ranging from 2-fold to 10-fold. 37,38 These mutations were selected to increase affinity through improved cross-strand interactions on the β-sheet and through optimized electrostatic interactions near the aromatic cage. Combining several of these mutations, if additive, would result in an improved affinity in the nanomolar range, suitable for many biochemical assays as a detection reagent.…”