Molecular dynamics simulation on HP1 protein binding by histone H3 tail methylation and phosphorylation

Trimethylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging, and heterochromatin formation. Phosphorylation of histone H3 has been linked with mitotic chromatin condensation. During mitosis in vivo, H3 lysine 9 methylation and serine 10 phosphorylation can occur concomitantly on the same histone tail, whereas the influence of phosphorylation to trimethylation H3 tail recruiting HP1 remains controversial. In this work, molecular dynamics simulation of HP1 complexed with both trimethylated and phosphorylated H3 tail were performed and compared with the results from the previous methylated H3‐HP1 trajectory. It is clear from the 10‐ns dynamics simulation that two adjacent posttranslational modifications directly increase the flexibility of the H3 tail and weaken HP1 binding to chromatin. A combinatorial readout of two adjacent posttranslational modifications—a stable methylation and a dynamic phosphorylation mark—establish a regulatory mechanism of protein–protein interactions. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

Genetically encoded fluorescent reporters of histone methylation in living cells

We report the design and characterization of two genetically encoded fluorescent reporters of histone protein methylation. The reporters are four-part chimeric proteins consisting of a substrate peptide from the N-terminus of histone H3 fused to a chromodomain (a natural methyllysine-specific recognition domain), sandwiched between a fluorescence resonance energy transfer (FRET)-capable pair of fluorophores, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Enzymatic methylation by a methyltransferase induces complexation of the methylated substrate peptide to the chromodomain, changing the FRET level between the flanking CFP and YFP domains. Reporters developed using the chromodomains from HP1 and Polycomb respond to enzymatic methylation at the lysine 9 and lysine 27 positions of histone H3, respectively, giving 60% and 28% YFP/CFP emission ratio increases in vitro or in single living cells. These reporters should be useful for studying gene silencing and X-chromosome inactivation with high spatial and temporal resolution in intact cells and may also aid in the search for conjectured histone demethylase activity.     

A quantitative analysis of histone methylation and acetylation isoforms from their deuteroacetylated derivatives: application to a series of knockout mutants

The core histones, H2A, H2B, H3 and H4, undergo post‐translational modifications (PTMs) including lysine acetylation, methylation and ubiquitylation, arginine methylation and serine phosphorylation. Lysine residues may be mono‐, di‐ and trimethylated, the latter resulting in an addition of mass to the protein that differs from acetylation by only 0.03639 Da, but that can be distinguished either on high‐performance mass spectrometers with sufficient mass accuracy and mass resolution or via retention times. Here we describe the use of chemical derivatization to quantify methylated and acetylated histone isoforms by forming deuteroacetylated histone derivatives prior to tryptic digestion and bottom‐up liquid chromatography‐mass spectrometric analysis. The deuteroacetylation of unmodified or mono‐methylated lysine residues produces a chemically identical set of tryptic peptides when comparing the unmodified and modified versions of a protein, making it possible to directly quantify lysine acetylation. In this work, the deuteroacetylation technique is used to examine a single histone H3 peptide with methyl and acetyl modifications at different lysine residues and to quantify the relative abundance of each modification in different deacetylase and methylase knockout yeast strains. This application demonstrates the use of the deuteroacetylation technique to characterize modification ‘cross‐talk’ by correlating different PTMs on the same histone tail. Copyright © 2013 John Wiley & Sons, Ltd.     

Quantitative chemical proteomics approach to identify post-translational modification-mediated protein-protein interactions

Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein-protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein-protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein-protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 N-terminus (H3K4Me(3)), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me(3). This study indicates that our cross-linking-assisted and SILAC-based protein identification (CLASPI) approach can be used to profile protein-protein interactions mediated by PTMs, such as lysine methylation.     

Influence of N-methylation on a cation-pi interaction produces a remarkably stable beta-hairpin peptide

The methylation of lysine in histone tails is a common posttranslational modification that functions in histone-regulated chromatin condensation, with binding of methylated lysine occurring in aromatic pockets on chromodomain proteins. We have synthesized a highly stable 12-residue beta-hairpin peptide that exploits the histone-related cation-pi interaction between a methylated lysine residue and a tryptophan residue. Thermodynamic analysis reveals significant entropic stabilization of the peptide due to methylation of the lysine residue. Chemical denaturation of the peptide demonstrates two-state behavior. In comparison to other reported, highly stable designed beta-hairpins, this peptide is the most thermally stable beta-hairpin reported to date. This study provides insight into the role of Lys methylation in histone proteins and more generally in mediating protein-protein interactions.     

LSD1: more than demethylation of histone lysine residues

Lysine-specific histone demethylase 1 (LSD1) represents the first example of an identified nuclear protein with histone demethylase activity. In particular, it plays a special role in the epigenetic regulation of gene expression, as it removes methyl groups from mono- and dimethylated lysine 4 and/or lysine 9 on histone H3 (H3K4me1/2 and H3K9me1/2), behaving as a repressor or activator of gene expression, respectively. Moreover, it has been recently found to demethylate monomethylated and dimethylated lysine 20 in histone H4 and to contribute to the balance of several other methylated lysine residues in histone H3 (i.e., H3K27, H3K36, and H3K79). Furthermore, in recent years, a plethora of nonhistone proteins have been detected as targets of LSD1 activity, suggesting that this demethylase is a fundamental player in the regulation of multiple pathways triggered in several cellular processes, including cancer progression. In this review, we analyze the molecular mechanism by which LSD1 displays its dual effect on gene expression (related to the specific lysine target), placing final emphasis on the use of pharmacological inhibitors of its activity in future clinical studies to fight cancer.Subject terms: Epigenetics, Histone post-translational modifications     

Traceless Synthesis of Asymmetrically Modified Bivalent Nucleosomes

Nucleosomes carry extensive post‐translational modifications (PTMs), which results in complex modification patterns that are involved in epigenetic signaling. Although two copies of each histone coexist in a nucleosome, they may not carry the same PTMs and are often differently modified (asymmetric). In bivalent domains, a chromatin signature prevalent in embryonic stem cells (ESCs), namely H3 methylated at lysine 4 (H3K4me3), coexists with H3K27me3 in asymmetric nucleosomes. We report a general, modular, and traceless method for producing asymmetrically modified nucleosomes. We further show that in bivalent nucleosomes, H3K4me3 inhibits the activity of the H3K27‐specific lysine methyltransferase (KMT) polycomb repressive complex 2 (PRC2) solely on the same histone tail, whereas H3K27me3 stimulates PRC2 activity across tails, thereby partially overriding the H3K4me3‐mediated repressive effect. To maintain bivalent domains in ESCs, PRC2 activity must thus be locally restricted or reversed.     

Structure and binding of the H4 histone tail and the effects of lysine 16 acetylation

The H4 histone tail plays a critical role in chromatin folding and regulation--it mediates strong interactions with the acidic patch of proximal nucleosomes and its acetylation at lysine 16 (K16) leads to partial unfolding of chromatin. The molecular mechanism associated with the H4 tail/acidic patch interactions and its modulation via K16 acetylation remains unknown. Here we employ a combination of molecular dynamics simulations, molecular docking calculations, and free energy computations to investigate the structure of the H4 tail in solution, the binding of the H4 tail with the acidic patch, and the effects of K16 acetylation. The H4 tail exhibits a disordered configuration except in the region Ala15-Lys20, where it exhibits a strong propensity for an α-helical structure. This α-helical region is found to dock very favorably into the acidic patch groove of a nucleosome with a binding free energy of approximately -7 kcal mol(-1). We have identified the specific interactions that stabilize this binding as well as the associated energetics. The acetylation of K16 is found to reduce the α-helix forming propensity of the H4 tail and K16's accessibility for mediating external interactions. More importantly, K16 acetylation destabilizes the binding of the H4 tail at the acidic patch by mitigating specific salt bridges and longer-ranged electrostatic interactions mediated by K16. Our study thus provides new microscopic insights into the compaction of chromatin and its regulation via posttranslational modifications of histone tails, which could be of interest to chromatin biology, cancer, epigenetics, and drug design.     

Structural study of a small molecule receptor bound to dimethyllysine in lysozyme

Lysine is a ubiquitous residue on protein surfaces. Post translational modifications of lysine, including methylation to the mono-, di- or trimethylated amine result in chemical and structural alterations that have major consequences for protein interactions and signalling pathways. Small molecules that bind to methylated lysines are potential tools to modify such pathways. To make progress in this direction, detailed structural data of ligands in complex with methylated lysine is required. Here, we report a crystal structure of p-sulfonatocalix[4]arene (sclx₄) bound to methylated lysozyme in which the lysine residues were chemically modified from Lys-NH₃ ⁺ to Lys-NH(Me₂)⁺. Of the six possible dimethyllysine sites, sclx₄ selected Lys116-Me₂ and the dimethylamino substituent was deeply buried in the calixarene cavity. This complex confirms the tendency for Lys-Me₂ residues to form cation–π interactions, which have been shown to be important in protein recognition of histone tails bearing methylated lysines. Supporting data from NMR spectroscopy and MD simulations confirm the selectivity for Lys116-Me₂ in solution. The structure presented here may serve as a stepping stone to the development of new biochemical reagents that target methylated lysines.     

NMR assignments and histone specificity of the ING2 PHD finger

The ING2 plant homeodomain (PHD) finger is recruited to the nucleosome through specific binding to histone H3 trimethylated at lysine 4 (H3K4me3). Here, we describe backbone and side chain assignments of the ING2 PHD finger, analyze its binding to the unmodified and modified histone and p53 peptides, and map the histone H3 and H3K4me3 binding sites based on chemical shift perturbation analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Isoxazole‐Derived Amino Acids are Bromodomain‐Binding Acetyl‐Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3

A range of isoxazole‐containing amino acids was synthesized that displaced acetyl‐lysine‐containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4‐mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole‐containing peptides are comparable to those of a hyperacetylated histone H4‐mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole‐based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4‐mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl‐lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3.     

Dioxygen Binding in the Active Site of Histone Demethylase JMJD2A and the Role of the Protein Environment

JMJD2A catalyses the demethylation of di‐ and trimethylated lysine residues in histone tails and is a target for the development of new anticancer medicines. Mechanistic details of demethylation are yet to be elucidated and are important for the understanding of epigenetic processes. We have evaluated the initial step of histone demethylation by JMJD2A and demonstrate the dramatic effect of the protein environment upon oxygen binding using quantum mechanics/molecular mechanics (QM/MM) calculations. The changes in electronic structure have been studied for possible spin states and different conformations of O₂, using a combination of quantum and classical simulations. O₂ binding to this histone demethylase is computed to occur preferentially as an end‐on superoxo radical bound to a high‐spin ferric centre, yielding an overall quintet ground state. The favourability of binding is strongly influenced by the surrounding protein: we have quantified this effect using an energy decomposition scheme into electrostatic and dispersion contributions. His182 and the methylated lysine assist while Glu184 and the oxoglutarate cofactor are deleterious for O₂ binding. Charge separation in the superoxo‐intermediate benefits from the electrostatic stabilization provided by the surrounding residues, stabilizing the binding process significantly. This work demonstrates the importance of the extended protein environment in oxygen binding, and the role of energy decomposition in understanding the physical origin of binding/recognition.     

A Genetically Encoded Allysine for the Synthesis of Proteins with Site‐Specific Lysine Dimethylation

Using the amber suppression approach, N^ϵ‐(4‐azidobenzoxycarbonyl)‐δ,ϵ‐dehydrolysine, an allysine precursor is genetically encoded in E. coli. Its genetic incorporation followed by two sequential biocompatible reactions allows convenient synthesis of proteins with site‐specific lysine dimethylation. Using this approach, dimethyl‐histone H3 and p53 proteins have been synthesized and used to probe functions of epigenetic enzymes including histone demethylase LSD1 and histone acetyltransferase Tip60. We confirmed that LSD1 is catalytically active toward H3K4me2 and H3K9me2 but inert toward H3K36me2, and methylation at p53 K372 directly activates Tip60 for its catalyzed acetylation at p53 K120.     

Genetically directing ɛ-N, N-dimethyl-L-lysine in recombinant histones

A molecular understanding of the biological phenomena orchestrated by lysine N(?)-methylation is impeded by the challenge of producing site-specifically and quantitatively methylated histones. Here, we report a general method that combines genetic code expansion and chemoselective reactions, for the quantitative, site-specific installation of dimethyl-lysine in recombinant histones. We demonstrate the utility of our method by preparing H3K9me2 and show that this modified histone is specifically recognized by heterochromatin protein 1 beta. Extensions of the strategy reported here will allow a range of chemoselective reactions (which have been used for residue-selective, but not site-selective protein modification) to be leveraged for site-specific protein modification.     

A Versatile Approach for Site‐Specific Lysine Acylation in Proteins

Using amber suppression in coordination with a mutant pyrrolysyl‐tRNA synthetase‐tRNA^Pyl pair, azidonorleucine is genetically encoded in E. coli . Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a site‐specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su‐H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified post‐translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.     

A Fluorescence‐Based Supramolecular Tandem Assay for Monitoring Lysine Methyltransferase Activity in Homogeneous Solution

The demand for practical and convenient enzyme assays for histone lysine methyltransferases (HKMTs) emerges along with the rapid development of this young class of enzymes. A supramolecular reporter pair composed of p‐sulfonatocalix[4]arene (CX4) and the fluorescent dye lucigenin (LCG) has been used to monitor enzymatic trimethylation of lysine residues in peptide substrates. The assay affords a switch‐ON fluorescence response and operates in a continuous, real‐time, and label‐free fashion. The underlying working principle relies on the higher affinity of the macrocycle towards the trimethylated product of the enzymatic reaction as compared to the substrate, which allows the assay to be carried out in the product‐selective mode. The final product incorporates a trimethylammonium moiety, a known high‐affinity binding motif for CX4. Two substrates corresponding to the H3 N‐terminal tail, namely, S2 (RTKQTA RKSTG GKAP) and S6 (QTA RKSTG GS), were selected as model compounds for methylation with the Neurospora crassa Dim‐5 enzyme and investigated by the newly developed supramolecular tandem HKMTs assay. Only the longer substrate S2 underwent methylation in solution. The potential of the assay for inhibitor screening was demonstrated by means of inhibition studies with 1,10‐phenanthroline to afford an inhibition constant of (70±20) μM .     

Specificity analysis-based identification of new methylation targets of the SET7/9 protein lysine methyltransferase

We applied peptide array methylation to determine an optimized target sequence for the SET7/9 (KMT7) protein lysine methyltransferase. Based on this, we identified 91 new peptide substrates from human proteins, many of them better than known substrates. We confirmed methylation of corresponding protein domains in?vitro and in?vivo with a high success rate for strongly methylated peptides and showed methylation of nine nonhistone proteins (AKA6, CENPC1, MeCP2, MINT, PPARBP, ZDH8, Cullin1, IRF1, and [weakly] TTK) and of H2A and H2B, which more than doubles the number of known SET7/9 targets. SET7/9 is inhibited by phosphorylation of histone and nonhistone substrate proteins. One lysine in the MINT protein is dimethylated in?vitro and in?vivo demonstrating that the product pattern created by SET7/9 depends on the amino acid sequence context of the target site.     

Thermodynamic consequences of Tyr to Trp mutations in the cation–π-mediated binding of trimethyllysine by the HP1 chromodomain

Evolution has converged on cation–π interactions for recognition of quaternary alkyl ammonium groups such as trimethyllysine (Kme3). While computational modelling indicates that Trp provides the strongest cation–π interaction of the native aromatic amino acids, there is limited corroborative data from measurements within proteins. Herein we investigate a Tyr to Trp mutation in the binding pocket of the HP1 chromodomain, a reader protein that recognizes Kme3. Binding studies demonstrate that the Trp-mediated cation–π interaction is about −5 kcal mol⁻¹ stronger, and the Y24W crystal structure shows that the mutation is not perturbing. Quantum mechanical calculations indicate that greater enthalpic binding is predominantly due to increased cation–π interactions. NMR studies indicate that differences in the unbound state of the Y24W mutation lead to enthalpy–entropy compensation. These results provide direct experimental quantification of Trp versus Tyr in a cation–π interaction and afford insight into the conservation of aromatic cage residues in Kme3 reader domains.

In this work, we experimentally validate that tryptophan provides the strongest cation–π binding interaction among aromatic amino acids and also lend insight into the importance of residue identity in trimethyllysine recognition by reader proteins.