Aromatic Cage Research Articles

Evolutionary convergence and divergence in archaeal chromosomal proteins and Chromo-like domains from bacteria and eukaryotes

SH3-fold-β-barrel domains of the chromo-like superfamily recognize epigenetic marks in eukaryotic proteins. Their provenance has been placed either in archaea, based on apparent structural similarity to chromatin-compacting Sul7d and Cren7 proteins, or in bacteria based on the presence of sequence homologs. Using sequence and structural evidence we establish that the archaeal Cren7/Sul7 proteins emerged from a zinc ribbon (ZnR) ancestor. Further, we show that the ancestral eukaryotic chromo-like domains evolved from bacterial versions, likely acquired from early endosymbioses, which already possessed an aromatic cage for recognition of modified amino-groups. These bacterial versions are part of a radiation of secreted SH3-fold domains, which spawned both chromo-like domains and classical SH3 domains in the context of peptide-recognition in the peptidoglycan or the extracellular matrix. This establishes that Cren7/Sul7 converged to a “SH3”-like state from a ZnR precursor via the loss of metal-chelation and acquisition of stronger hydrophobic interactions; it is unlikely to have participated in the evolution of the chromo-like domains. We show that archaea possess several Cren7/Sul7-related proteins with intact Zn-chelating ligands, which we predict to play previously unstudied roles in chromosome segregation during cell-division comparable to the PRC barrel and CdvA domain proteins.

The molecular selectivity of UNC3866 inhibitor for Polycomb CBX7 protein from molecular dynamics simulation

Polycomb CBX proteins regulate gene expression by targeting Polycomb repressive complex 1 (PRC1) to sites of H3K27me3 via their chromodomains, which plays a key role in the development of numerous cancers. UNC3866, is a recently reported peptide-based inhibitor of the methyllysine (Kme) reading function of CBX chromodomains (CBX2, 4 and 6–8). The previous experiments showed that UNC3866 bound the chromodomains of CBX7 strongly, with ∼20-fold selectivity over other CBX chromodomains. However, the potential mechanism of UNC3866 preferentially binding to CBX7 is still unknown. In this study, we performed two pairs of microsecond molecular dynamic simulations (CBX2 (-UNC3866)) and (CBX7 (-UNC3866)) to study the inhibition and isoform-selective mechanism of UNC3866 to CBX7. The conformational analysis of apo- and holo- CBX2 and CBX7 indicated that the aromatic cage of CBX7 protein was more prone to be induced by UNC3866 relative to CBX2 protein. The results of predicted binding free energy suggested the binding affinity of UNC3866 with CBX7 was stronger than that with CBX2, because of the lower binding free energy of the former. Furthermore, the energetic origin of UNC3866 selective for CBX7 protein mainly came from the higher van der Waals contributions. The binding mode analysis showed that Asn47 of CBX2 formed a hydrogen bond with the OH group of C-terminal cap of UNC3866, inducing the conformational changes of diethyllysine of UNC3866 that is obviously different from that in CBX7. Additionally, His39 in CBX2 chromodomain interrupted the structured aromatic cage, partly explaining the reason for UNC3866 preferring for binding to CBX7. The proposal of this selective mechanism could be helpful for the rational design of novel selective inhibitors of the Polycomb CBX protein.

One-pot Annulation for Biaryl-fused Monocarba-closo-dodecaborate through Aromatic B-H Bond Disconnection.

We have developed a one-pot annulation reaction of monocarba-closo-dodecaborate with cyclic diaryliodonium salts to afford biaryl-fused derivatives. Aryl functionalities are introduced at both the 1-carbon and unreactive ortho-boron vertices of the "σ-aromatic" carborane cage without the need for pre-functionalization. DFT calculations revealed that the palladium-catalyzed C-B bond-formation step in this process proceeds through a concerted metalation-deprotonation (CMD)-type pathway for the B-H bond disconnection on the aromatic cage, though such bonds are generally regarded as hydridic.

Asymmetric synthesis, molecular modeling and biological evaluation of 5-methyl-3-aryloxazolidine-2,4-dione enantiomers as monoamine oxidase (MAO) inhibitors

Single enantiomers of the new 5-methyl-3-aryloxazolidine-2,4-diones have been obtained either by an asymmetric synthesis using the chiral pool strategy or by a semipreparative resolution of the racemic compound by HPLC on an optically active stationary phase. The single enantiomers were assayed for their in vitro monoamine oxidase (hMAO) inhibitory activity and selectivity. The most potent inhibitor among the studied compounds has been found as (5R)-3-phenyl-5-methyl-2,4-oxazolidinedione (compound 1-R) which appeared to be a good antidepressant drug candidate since it inhibited hMAO-A selectively, competitively and reversibly with Ki values in the micromolar range (0.16 ± 0.01 μM). To better understand the enzyme-inhibitor interaction and to explain the efficiency and selectivity of the compounds toward hMAOs, molecular modeling studies were carried out on new, high resolution hMAO-A and hMAO-B crystallographic structures. According to binding energies and inhibition constants obtained from molecular docking calculations, compound 1-R has been found as the most selective MAO-A inhibitor and its weak binding affinities to MAO-B (large Ki values) led to the enhancement in MAO-A selectivity. It bounded in close proximity to FAD in the active site of MAO-A and situated near the aromatic cage by means of π-alkyl interactions with Tyr407 and Phe352 whereas its position in MAO-B was 10 Å far from FAD and it was situated outside the Ile199 gate of the active site. None of the studied compounds showed any cytototoxicity on HepG2 cells at 1 and 5 µM concentrations.

Charging nanoparticles: increased binding of Gd@C82(OH)22 derivatives to human MMP-9.

Unlike most matrix metalloproteinase (MMP) inhibitors, which target the conserved catalytic zinc site, Gd@C82(OH)22 indirectly inhibits MMP-9 activity by binding at the ligand specificity S1' loop. The allosteric binding makes Gd@C82(OH)22 a promising inhibitor selective for MMP-9. However, the hydrophobic nature of the aromatic carbon cage may cause Gd@C82(OH)22 to self-aggregate in aqueous solutions, hence weakening the binding. In this study, we designed Gd@C82(OH)22 derivatives aiming at improving the binding affinity for MMP-9. Upon a mutation that substitutes a new functional group (-PO42-, -CH2CO2-, -CO2-, -NH3+, or -CONH2) for a hydroxyl group on the fullerenol surface, we calculated the changes in the binding free energy to the catalytic domain of human MMP-9 using the free energy perturbation (FEP) method. We found that the higher the net charge of the functional group, the stronger the binding. Compared with Gd@C82(OH)22, Gd@C82(OH)21(PO4)2- binds at least 1.5 to 2.5 kcal mol-1 more strongly to MMP-9. The binding is specifically controlled by electrostatic interactions between the phosphate group and the charged residues at the binding site. In addition to the net charge, the binding free energy can be delicately adjusted by other factors, such as the functionalization site on Gd@C82(OH)22, the local environment of the putative binding site of MMP-9, and the presence of ions near the charged functional group. The results of our study shed light on the potential of developing Gd@C82(OH)22 derivatives as nanodrugs for treating the pathological diseases associated with unregulated MMP-9 activity.

Conformational Selection of Dimethylarginine Recognition by the Survival Motor Neuron Tudor Domain.

Tudor domains bind to dimethylarginine (DMA) residues, which are post-translational modifications that play a central role in gene regulation in eukaryotic cells. NMR spectroscopy and quantum calculations are combined to demonstrate that DMA recognition by Tudor domains involves conformational selection. The binding mechanism is confirmed by a mutation in the aromatic cage that perturbs the native recognition mode of the ligand. General mechanistic principles are delineated from the combined results, indicating that Tudor domains utilize cation-π interactions to achieve ligand recognition.

Conformational Selection of Dimethylarginine Recognition by the Survival Motor Neuron Tudor Domain

AbstractTudor domains bind to dimethylarginine (DMA) residues, which are post‐translational modifications that play a central role in gene regulation in eukaryotic cells. NMR spectroscopy and quantum calculations are combined to demonstrate that DMA recognition by Tudor domains involves conformational selection. The binding mechanism is confirmed by a mutation in the aromatic cage that perturbs the native recognition mode of the ligand. General mechanistic principles are delineated from the combined results, indicating that Tudor domains utilize cation–π interactions to achieve ligand recognition.

H3K14ac is linked to methylation of H3K9 by the triple Tudor domain of SETDB1

SETDB1 is an essential H3K9 methyltransferase involved in silencing of retroviruses and gene regulation. We show here that its triple Tudor domain (3TD) specifically binds to doubly modified histone H3 containing K14 acetylation and K9 methylation. Crystal structures of 3TD in complex with H3K14ac/K9me peptides reveal that peptide binding and K14ac recognition occurs at the interface between Tudor domains (TD) TD2 and TD3. Structural and biochemical data demonstrate a pocket switch mechanism in histone code reading, because K9me1 or K9me2 is preferentially recognized by the aromatic cage of TD3, while K9me3 selectively binds to TD2. Mutations in the K14ac/K9me binding sites change the sub-nuclear localization of 3TD. ChIP-seq analyses show that SETDB1 is enriched at H3K9me3 regions and K9me3/K14ac is enriched at SETDB1 binding sites overlapping with LINE elements, suggesting that recruitment of the SETDB1 complex to K14ac/K9me regions has a role in silencing of active genomic regions.

Investigation of Trimethyllysine Binding by the HP1 Chromodomain via Unnatural Amino Acid Mutagenesis.

Trimethyllysine (Kme3) reader proteins are targets for inhibition due to their role in mediating gene expression. Although all such reader proteins bind Kme3 in an aromatic cage, the driving force for binding may differ; some readers exhibit evidence for cation-π interactions whereas others do not. We report a general unnatural amino acid mutagenesis approach to quantify the contribution of individual tyrosines to cation binding using the HP1 chromodomain as a model system. We demonstrate that two tyrosines (Y24 and Y48) bind to a Kme3-histone tail peptide via cation-π interactions, but linear free energy trends suggest they do not contribute equally to binding. X-ray structures and computational analysis suggest that the distance and degree of contact between Tyr residues and Kme3 plays an important role in tuning cation-π-mediated Kme3 recognition. Although cation-π interactions have been studied in a number of proteins, this work is the first to utilize direct binding assays, X-ray crystallography, and modeling, to pinpoint factors that influence the magnitude of the individual cation-π interactions.

GyrI-like proteins catalyze cyclopropanoid hydrolysis to confer cellular protection

GyrI-like proteins are widely distributed in prokaryotes and eukaryotes, and recognized as small-molecule binding proteins. Here, we identify a subfamily of these proteins as cyclopropanoid cyclopropyl hydrolases (CCHs) that can catalyze the hydrolysis of the potent DNA-alkylating agents yatakemycin (YTM) and CC-1065. Co-crystallography and molecular dynamics simulation analyses reveal that these CCHs share a conserved aromatic cage for the hydrolytic activity. Subsequent cytotoxic assays confirm that CCHs are able to protect cells against YTM. Therefore, our findings suggest that the evolutionarily conserved GyrI-like proteins confer cellular protection against diverse xenobiotics via not only binding, but also catalysis.

A Unique pH-Dependent Recognition of Methylated Histone H3K4 by PPS and DIDO

The protein partner of Sans-fille (PPS) and its human homolog DIDO mediate diverse chromatin activities, including the regulation of stemness genes in embryonic stem cells and splicing in Drosophila. Here, we show that the PHD fingers of PPS and DIDO recognize the histone mark H3K4me3 in a pH-dependent manner: the binding is enhanced at high pH values but is decreased at low pH. Structural analysis reveals that the pH dependency is due to the presence of a histidine residue in the K4me3-binding aromatic cage of PPS. The pH-dependent mechanism is conserved in DIDO but is lost in yeast Bye1. Acidification of cells leads to the accelerated efflux of endogenous DIDO, indicating the pH-dependent sensing of H3K4me3 invivo. This novel mode for the recognition of H3K4me3 establishes the PHD fingers of PPS and DIDO as unique epigenetic readers and high pH sensors and suggests a role for the histidine switch during mitosis.

Recognition dynamics of dopamine to human Monoamine oxidase B: role of Leu171/Gln206 and conserved water molecules in the active site cavity

The human Monoamine oxidase (hMAO) metabolizes several biogenic amine neurotransmitters and is involved in different neurological disorders. Extensive MD simulation studies of dopamine-docked hMAO B structures have revealed the stabilization of amino-terminal of the substrate by a direct and water-mediated interaction of catalytic tyrosines, Gln206, and Leu171 residues. The catechol ring of the substrate is stabilized by Leu171(C–H)⋯π(Dop)⋯(H–C) Ile199 interaction. Several conserved water molecules are observed to play a role in the recognition of substrate to the enzyme, where W1 and W2 associate in dopamine– FAD interaction, reversible dynamics of W3 and W4 influenced the coupling of Tyr435 to Trp432 and FAD, and W5 and W8 stabilized the catalytic Tyr188/398 residues. The W6, W7, and W8 water centers are involved in the recognition of catalytic residues and FAD with the N+- site of dopamine through hydrogen bonding interaction. The recognition of substrate to gating residues is made through W9, W10, and W11 water centers. Beside the interplay of water molecules, the catalytic aromatic cage has also been stabilized by π⋯water, π⋯C–H, and π⋯π interactions. The topology of conserved water molecular sites along with the hydration dynamics of catalytic residues, FAD, and dopamine has added a new feature on the substrate binding chemistry in hMAO B which may be useful for substrate analog inhibitor design.

Developing Spindlin1 small-molecule inhibitors by using protein microarrays.

The discovery of inhibitors of methyl- and acetyl-binding domains has provided evidence for the 'druggability' of epigenetic effector molecules. The small-molecule probe UNC1215 prevents methyl-dependent protein-protein interactions by engaging the aromatic cage of MBT domains and, with lower affinity, Tudor domains. Using a library of tagged UNC1215 analogs, we screened a protein-domain microarray of human methyllysine effector molecules to rapidly detect compounds with new binding profiles with either increased or decreased specificity. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor-domain-containing protein Spindlin1 (SPIN1). Structural studies facilitated the rational synthesis of SPIN1 inhibitors with increased selectivity (EML631-633), which engage SPIN1 in cells, block its ability to 'read' H3K4me3 marks and inhibit its transcriptional-coactivator activity. Protein microarrays can thus be used as a platform to 'target-hop' and identify small molecules that bind and compete with domain-motif interactions.

Discovery of Peptidomimetic Ligands of EED as Allosteric Inhibitors of PRC2.

The function of EED within polycomb repressive complex 2 (PRC2) is mediated by a complex network of protein-protein interactions. Allosteric activation of PRC2 by binding of methylated proteins to the embryonic ectoderm development (EED) aromatic cage is essential for full catalytic activity, but details of this regulation are not fully understood. EED's recognition of the product of PRC2 activity, histone H3 lysine 27 trimethylation (H3K27me3), stimulates PRC2 methyltransferase activity at adjacent nucleosomes leading to H3K27me3 propagation and, ultimately, gene repression. By coupling combinatorial chemistry and structure-based design, we optimized a low-affinity methylated jumonji, AT-rich interactive domain 2 (Jarid2) peptide to a smaller, more potent peptidomimetic ligand (Kd = 1.14 ± 0.14 μM) of the aromatic cage of EED. Our strategy illustrates the effectiveness of applying combinatorial chemistry to achieve both ligand potency and property optimization. Furthermore, the resulting ligands, UNC5114 and UNC5115, demonstrate that targeted disruption of EED's reader function can lead to allosteric inhibition of PRC2 catalytic activity.

Probing a General Rule towards Thermodynamic Stabilities of Mono BN-doped Lower Polyenes.

The BN-doped organic analogues are interesting as aliphatic amineboranes for hydrogen storage, precursors for aromatic borazines and adsorbent cage azaboranes. However, BN-doped aliphatic polyenes remained undeveloped. Herein, we perform theoretical calculations on two mono BN-doped aliphatic lower polyenes, 1,3-butadiene and 1,3,5-hexatriene. A general rule is proposed, i.e., isomers with terminal nitrogen and directly BN-connected, N-B(R), in particular, are of significant thermodynamic stability as compared with their inverse analogues (where boron is at the terminal position). The N-B(R) type isomers are found to be the most stable ones in both polyenes. Isomers with terminal B and N are of intermediate stability. Highly destabilized isomers are those with one terminal methylene group and one terminal heteroatom in the butadiene series, and two terminal methylene groups in the hexatriene series. Rules established here may lead researchers to synthesize isomers with particular thermodynamic stability.

Discovery and Molecular Basis of a Diverse Set of Polycomb Repressive Complex 2 Inhibitors Recognition by EED.

Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity.

Site-specific azide-acetyllysine photochemistry on epigenetic readers for interactome profiling.

Chemical modifications on DNA, RNA and histones are recognized by an array of 'reader' modules to regulate transcriptional programming and cell fate. However, identification of reader-specific interacting partners in a dynamic cellular environment remains a significant challenge. Herein, we report a chemoproteomic approach termed 'interaction-based protein profiling' (IBPP) to characterize novel interacting partners of potentially any reader protein. IBPP harnesses a photosensitive amino acid introduced into the hydrophobic pocket of a reader module to crosslink and enrich transient interacting partners that are inaccessible to traditional methods. Using bromodomain-containing protein 4 (BRD4) as a paradigm, we engineer an 'aromatic cage' of the bromodomain to introduce 4-azido-l-phenylalanine (pAzF) without compromising its ability to recognize acetylated lysine residues in histone proteins. We establish the binding efficiency, substrate specificity and crosslinking ability of the engineered 'reader' module in biochemical assays. Applying IBPP, we uncovered novel acetylated interacting partners of BRD4, such as transcription factors, expanding on its previously unappreciated role in diverse biological processes. By setting up an azide-acetyllysine photoreaction deep inside the bromodomain aromatic cage as a means to detect protein acetylation, our approach provides a potentially general platform for rapid and unbiased profiling of interacting partners of diverse epigenetic readers whose functions in eukaryotic gene regulation remain convoluted.

Molecular recognition at cholinergic synapses: acetylcholine versus choline.

Neuromuscular acetylcholine (ACh) receptors have a high affinity for the neurotransmitter ACh and a low affinity for its metabolic product choline. At each transmitter binding site three aromatic groups determine affinity, and together provide ∼50% more binding energy for ACh than for choline. Deprotonation of αY190 by a nearby lysine strengthens the interaction between this aromatic ring and both ACh and choline. H-bonds position ACh and choline differently in the aromatic cage to generate the different affinities. Acetylcholine (ACh) released at the vertebrate nerve-muscle synapse is hydrolysed rapidly to choline (Cho), so endplate receptors (AChRs) are exposed to high concentrations of both of these structurally related ligands. To understand how these receptors distinguish ACh and Cho, we used single-channel electrophysiology to measure resting affinities (binding free energies) of these and other agonists in adult-type mouse AChRs having a mutation(s) at the transmitter-binding sites. The aromatic rings of αY190, αW149 and αY198 each provide ∼50% less binding energy for Cho compared to ACh. At αY198 a phenylalanine substitution had no effect, but at αY190 this substitution caused a large, agonist-independent loss in binding energy that depended on the presence of αK145. The results suggest that (1) αY190 is deprotonated by αK145 to strengthen the interaction between this benzene ring and the agonist's quaternary ammonium (QA) and (2) AChRs respond strongly to ACh because an H-bond positions the QA to interact optimally with the rings, and weakly to Cho because a different H-bond tethers the ligand to misalign the QA and form weaker interactions with the aromatic groups. The results suggest that the difference in ACh versus Cho binding energies is determined by different ligand positions within a fixed protein structure.

Structural Analysis Provides Mechanistic Insight into Nicotine Oxidoreductase from Pseudomonas putida.

The first structure of nicotine oxidoreductase (NicA2) was determined by X-ray crystallography. Pseudomonas putida has evolved nicotine-degrading activity to provide a source of carbon and nitrogen. The structure establishes NicA2 as a member of the monoamine oxidase family. Residues 1-50 are disordered and may play a role in localization. The nicotine-binding site proximal to the isoalloxazine ring of flavin shows an unusual composition of the classical aromatic cage (W427 and N462). The active site architecture is consistent with the proposed binding of the deprotonated form of the substrate and the flavin-dependent oxidation of the pyrrolidone C-N bond followed by nonenzymatic hydrolysis.

Empirical Valence Bond Simulations of the Hydride-Transfer Step in the Monoamine Oxidase A Catalyzed Metabolism of Noradrenaline.

Monoamine oxidases (MAOs) A and B are flavoenzymes responsible for the metabolism of biogenic amines, such as dopamine, serotonin, and noradrenaline (NA), which is why they have been extensively implicated in the etiology and course of various neurodegenerative disorders and, accordingly, used as primary pharmacological targets to treat these debilitating cognitive diseases. The precise chemical mechanism through which MAOs regulate the amine concentration, which is vital for the development of novel inhibitors, is still not unambiguously determined in the literature. In this work, we present atomistic empirical valence bond simulations of the rate-limiting step of the MAO-A-catalyzed NA (norepinephrine) degradation, involving hydride transfer from the substrate α-methylene group to the flavin moiety of the flavin adenine dinucleotide prosthetic group, employing the full dimensionality and thermal fluctuations of the hydrated enzyme, with extensive configurational sampling. We show that MAO-A lowers the free energy of activation by 14.3 kcal mol-1 relative to that of the same reaction in aqueous solution, whereas the calculated activation free energy of ΔG‡ = 20.3 ± 1.6 kcal mol-1 is found to be in reasonable agreement with the correlated experimental value of 16.5 kcal mol-1. The results presented here strongly support the fact that both MAO-A and MAO-B isoforms function by the same hydride-transfer mechanism. We also considered a few point mutations of the "aromatic cage" tyrosine residue (Tyr444Phe, Tyr444Leu, Tyr444Trp, Tyr444His, and Tyr444Glu), and the calculated changes in the reaction barriers are in agreement with the experimental values, thus providing further support to the proposed mechanism.