Evaluation of acyllysine isostere interactions with the aromatic pocket of the AF9 YEATS domain

Amide−π interactions, in which an amide interacts with an aromatic group, are ubiquitous in biology, yet remain understudied relative to other noncovalent interactions. Recently, we demonstrated that an electrostatically tunable amide−π interaction is key to recognition of histone acyllysine by the AF9 YEATS domain, a reader protein which has emerged as a therapeutic target due to its dysregulation in cancer. Amide isosteres are commonly employed in drug discovery, often to prevent degradation by proteases, and have proven valuable in achieving selectivity when targeting epigenetic proteins. However, like amide−π interactions, interactions of amide isosteres with aromatic rings have not been thoroughly studied despite widespread use. Herein, we evaluate the recognition of a series of amide isosteres by the AF9 YEATS domain using genetic code expansion to evaluate the amide isostere−π interaction. We show that compared to the amide−π interaction with the native ligand, each isostere exhibits similar electrostatic tunability with an aromatic residue in the binding pocket, demonstrating that the isosteres maintain similar interactions with the aromatic residue. We identify a urea-containing ligand that binds with enhanced affinity for the AF9 YEATS domain, offering a promising starting point for inhibitor development. Furthermore, we demonstrate that carbamate and urea isosteres of crotonyllysine are resistant to enzymatic removal by SIRT1, a protein that cleaves acyl post-translational modifications, further indicating the potential of amide isosteres in YEATS domain inhibitor development. These results also provide experimental precedent for interactions of these common drug discovery moieties with aromatic rings that can inform computational methods.     

Targeting disordered-structured domain interactions in Galectin-3 based on NMR and enhanced MD

Intrinsically disordered regions (IDRs) are common and important functional domains in many proteins. However, IDRs are difficult to target for drug development due to the lack of defined structures that would facilitate the identification of possible drug-binding pockets. Galectin-3 is a carbohydrate-binding protein of which overexpression has been implicated in a wide variety of disorders, including cancer and inflammation. Apart from its carbohydrate-recognition/binding domain (CRD), Galectin-3 also contains a functionally important disordered N-terminal domain (NTD) that contacts the C-terminal domain (CTD) and could be a target for drug development. To overcome challenges involved in inhibitor design due to lack of structure and the highly dynamic nature of the NTD, we used a protocol combining nuclear magnetic resonance data from recombinant Galectin-3 with accelerated molecular dynamics (MD) simulations. This approach identified a pocket in the CTD with which the NTD makes frequent contact. In accordance with this model, mutation of residues L131 and L203 in this pocket caused loss of Galectin-3 agglutination ability, signifying the functional relevance of the cavity. In silico screening was used to design candidate inhibitory peptides targeting the newly discovered cavity, and experimental testing of only three of these yielded one peptide that inhibits the agglutination promoted by wild-type Galectin-3. NMR experiments further confirmed that this peptide indeed binds to a cavity in the CTD, not within the actual CRD. Our results show that it is possible to apply a combination of MD simulations and NMR experiments to precisely predict the binding interface of a disordered domain with a structured domain, and furthermore use this predicted interface for designing inhibitors. This procedure can potentially be extended to many other targets in which similar IDR interactions play a vital functional role.     

Probing the interaction between cHAVc3 peptide and the EC1 domain of E-cadherin using NMR and molecular dynamics simulations

The goal of this work is to probe the interaction between cyclic cHAVc3 peptide and the EC1 domain of human E-cadherin protein. Cyclic cHAVc3 peptide (cyclo(1,6)Ac-CSHAVC-NH₂) binds to the EC1 domain as shown by chemical shift perturbations in the 2D ¹H,-¹⁵N-HSQC NMR spectrum. The molecular dynamics (MD) simulations of the EC1 domain showed folding of the C-terminal tail region into the main head region of the EC1 domain. For cHAVc3 peptide, replica exchange molecular dynamics (REMD) simulations generated five structural clusters of cHAVc3 peptide. Representative structures of cHAVc3 and the EC1 structure from MD simulations were used in molecular docking experiments with NMR constraints to determine the binding site of the peptide on EC1. The results suggest that cHAVc3 binds to EC1 around residues Y36, S37, I38, I53, F77, S78, H79, and I94. The dissociation constants (K_d values) of cHAVc3 peptide to EC1 were estimated using the NMR chemical shifts data and the estimated K_ds are in the range of .5 × 10^?5–7.0 × 10^?5 M.     

Rifampicin-independent interactions between the pregnane X receptor ligand binding domain and peptide fragments of coactivator and corepressor proteins

The pregnane X receptor (PXR), a member of the nuclear receptor superfamily, regulates the expression of drug-metabolizing enzymes in a ligand-dependent manner. The conventional view of nuclear receptor action is that ligand binding enhances the receptor's affinity for coactivator proteins, while decreasing its affinity for corepressors. To date, however, no known rigorous biophysical studies have been conducted to investigate the interaction among PXR, its coregulators, and ligands. In this work, steady-state total internal reflection fluorescence microscopy (TIRFM) and total internal reflection with fluorescence recovery after photobleaching were used to measure the thermodynamics and kinetics of the interaction between the PXR ligand binding domain and a peptide fragment of the steroid receptor coactivator-1 (SRC-1) in the presence and absence of the established PXR agonist, rifampicin. Equilibrium dissociation and dissociation rate constants of ~5 μM and ~2 s(-1), respectively, were obtained in the presence and absence of rifampicin, indicating that the ligand does not enhance the affinity of the PXR and SRC-1 fragments. Additionally, TIRFM was used to examine the interaction between PXR and a peptide fragment of the corepressor protein, the silencing mediator for retinoid and thyroid receptors (SMRT). An equilibrium dissociation constant of ~70 μM was obtained for SMRT in the presence and absence of rifampicin. These results strongly suggest that the mechanism of ligand-dependent activation in PXR differs significantly from that seen in many other nuclear receptors.     

Hotspot mutations in the structured ENL YEATS domain link aberrant transcriptional condensates and cancer

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Modeling and prediction of binding affinities between the human amphiphysin SH3 domain and its peptide ligands using genetic algorithm-Gaussian processes

In this article, we discuss the application of the Gaussian process (GP) and other statistical methods (PLS, ANN, and SVM) for the modeling and prediction of binding affinities between the human amphiphysin SH3 domain and its peptide ligands. Divided physicochemical property scores of amino acids, involving significant hydrogen bond, electronic, hydrophobic, and steric properties, was used to characterize the peptide structures, and quantitative structure-affinity relationship models were then constructed by PLS, ANN, SVM, and GP coupled with genetic algorithm-variable selection. The results show that: (i) since the significant flexibility and high complexity possessed in polypeptide structures, linear PLS method was incapable of fulfilling a satisfying behavior on SH3 domain binding peptide dataset; (ii) the overfitting involved in training process has decreased the predictive power of ANN model to some extent; (iii) both SVM and GP have a good performance for SH3 domain binding peptide dataset. Moreover, by combining linear and nonlinear terms in the covariance function, the GP is capable of handling linear and nonlinear-hybrid relationship, and which thus obtained a more stable and predictable model than SVM. Analyses of GP models showed that diversified properties contribute remarkable effect to the interactions between the SH3 domain and the peptides. Particularly, steric property and hydrophobicity of P(2), electronic property of P(0), and electronic property and hydrogen bond property of P(-3) in decapeptide (P(4)P(3)P(2)P(1)P(0)P(-1)P(-2)P(-3)P(-4)P(-5)) significantly contribute to the binding affinities of SH3 domain-peptide interactions.     

Salt-dependent intra- and internucleosomal interactions of the H3 tail domain in a model oligonucleosomal array

The core histone tail domains are known to be key regulators of chromatin structure and function. The tails are required for condensation of nucleosome arrays into secondary and tertiary chromatin structures, yet little is known regarding tail structures or sites of tail interactions in chromatin. We have developed a system to test the hypothesis that the tails participate in internucleosomal interactions during salt-dependent chromatin condensation, and here we used it to examine interactions of the H3 tail domain. We found that the H3 tail participates primarily in intranucleosome interactions when the nucleosome array exists in an extended "beads-on-a-string" conformation and that tail interactions reorganize to engage in primarily internucleosome interactions as the array successively undergoes salt-dependent folding and oligomerization. These results indicated that the location and interactions of the H3 tail domain are dependent upon the degree of condensation of the nucleosomal array, suggesting a mechanism by which alterations in tail interactions may elaborate different structural and functional states of chromatin.     

The H3 tail domain participates in multiple interactions during folding and self-association of nucleosome arrays

The core histone tail domains play a central role in chromatin structure and epigenetic processes controlling gene expression. Although little is known regarding the molecular details of tail interactions, it is likely that they participate in both short-range and long-range interactions between nucleosomes. Previously, we demonstrated that the H3 tail domain participates in internucleosome interactions during MgCl(2)-dependent condensation of model nucleosome arrays. However, these studies did not distinguish whether these internucleosome interactions represented short-range intra-array or longer-range interarray interactions. To better understand the complex interactions of the H3 tail domain during chromatin condensation, we have developed a new site-directed cross-linking method to identify and quantify interarray interactions mediated by histone tail domains. Interarray cross-linking was undetectable under salt conditions that induced only local folding, but was detected concomitant with salt-dependent interarray oligomerization at higher MgCl(2) concentrations. Interestingly, lysine-to-glutamine mutations in the H3 tail domain to mimic acetylation resulted in little or no reduction in interarray cross-linking. In contrast, binding of a linker histone caused a much greater enhancement of interarray interactions for unmodified H3 tails compared to "acetylated" H3 tails. Collectively these results indicate that H3 tail domain performs multiple functions during chromatin condensation via distinct molecular interactions that can be differentially regulated by acetylation or binding of linker histones.     

Endothelin and its receptor interactions: role of extracellular receptor domain and length of peptide ligands

Human endothelin B receptor and its domain-truncated forms were cloned and expressed in Pichia pastoris. Ligand binding studies with expressed proteins were carried out using biotinylated endothelins. Competitive binding and liposome incorporation studies showed that the extracellular region is essential for ligand binding and that longer peptides have higher affinity.     

Insights into the molecular interactions between aminopeptidase and amyloid beta peptide using molecular modeling techniques

Amyloid beta (Aβ) peptides play a central role in the pathogenesis of Alzheimer’s disease. The accumulation of Aβ peptides in AD brain was caused due to overproduction or insufficient clearance and defects in the proteolytic degradation of Aβ peptides. Hence, Aβ peptide degradation could be a promising therapeutic approach in AD treatment. Recent experimental report suggests that aminopeptidase from Streptomyces griseus KK565 (SGAK) can degrade Aβ peptides but the interactive residues are yet to be known in detail at the atomic level. Hence, we developed the three-dimensional model of aminopeptidase (SGAK) using SWISS-MODEL, Geno3D and MODELLER. Model built by MODELLER was used for further studies. Molecular docking was performed between aminopeptidase (SGAK) with wild-type and mutated Aβ peptides. The docked complex of aminopeptidase (SGAK) and wild-type Aβ peptide (1IYT.pdb) shows more stability than the other complexes. Molecular docking and MD simulation results revealed that the residues His93, Asp105, Glu139, Glu140, Asp168 and His255 are involved in the hydrogen bonding with Aβ peptide and zinc ions. The interactions between carboxyl oxygen atoms of Glu139 of aminopeptidase (SGAK) with water molecule suggest that the Glu139 may be involved in the nucleophilic attack on Ala2–Glu3 peptide bond of Aβ peptide. Hence, amino acid Glu139 of aminopeptidase (SGAK) might play an important role to degrade Aβ peptides, a causative agent of Alzheimer’s disease.     

Modulation of protein–ligand interactions by photocleavage of a cyclic peptide using phosphatidylinositol 3‐kinase SH3 domain as model system

To photomodulate the interaction of the phosphatidylinositol 3‐kinase SH3 domain with a peptide ligand, a cyclic peptide (cyclic‐1) with a photolabile side chain‐to‐side chain linker was synthesized. The conformation of cyclic‐1 differs from that of the parent linear peptide, but becomes identical by UV‐irradiation. Accordingly, the binding affinity of cyclic‐1 to the SH3 domain increased upon conversion of the cyclic to a linear flexible structure by irradiation (K_d: 3.4 ± 1.7 and 0.9 ± 0.3 mM , respectively). These results confirm the usefulness of a photocleavable peptide for photocontrol of peptide–protein interactions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Mapping peptide hormone-receptor interactions using a disulfide-trapping approach

Efforts to elucidate the nature of the bimolecular interaction of parathyroid hormone (PTH) with its cognate receptor, the PTH receptor type 1 (PTHR1), have relied heavily on benzoylphenylalanine- (Bpa-) based photoaffinity cross-linking. However, given the flexibility, size, and shape of Bpa, the resolution at the PTH-PTHR1 interface appears to be reaching the limit of this technique. Here we employ a disulfide-trapping approach developed by others primarily for use in screening compound libraries to identify novel ligands. In this method, cysteine substitutions are introduced into a specific site within the ligand and a region in the receptor predicted to interact with each other. Upon ligand binding, if these cysteines are in close proximity, they form a disulfide bond. Since the geometry governing disulfide bond formation is more constrained than Bpa cross-linking, this novel approach can be employed to generate a more refined molecular model of the PTH-PTHR1 complex. Using a PTH analogue containing a cysteine at position 1, we probed 24 sites and identified 4 in PTHR1 to which cross-linking occurred. Importantly, previous photoaffinity cross-linking studies using a PTH analogue with Bpa at position 1 only identified a single interaction site. The new sites identified by the disulfide-trapping procedure were used as constraints in molecular dynamics simulations to generate an updated model of the PTH-PTHR1 complex. Mapping by disulfide trapping extends and complements photoaffinity cross-linking. It is applicable to other peptide-receptor interfaces and should yield insights about yet unknown sites of ligand-receptor interactions, allowing for generation of more refined models.     

Ion-pair interactions between voltage-sensing domain IV and pore domain I regulate CaV1.1 gating

The voltage-gated calcium channel Ca_V1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of Ca_V1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire Ca_V1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for Ca_V1.1 and Ca_V1.2 L-type calcium channels. Moreover, in Ca_V1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-Ca_V1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V_1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-Ca_V1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V_1/2, whereas no further shift of V_1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in Ca_V1.1.     

Intermolecular interactions between the SH3 domain and the proline-rich TH region of Bruton's tyrosine kinase

The SH3 domain of Bruton's tyrosine kinase (Btk) is preceded by the Tec homology (TH) region containing proline-rich sequences. We have studied a protein fragment containing both the Btk SH3 domain and the proline-rich sequences of the TH region (PRR-SH3). Intermolecular NMR cross-relaxation measurements, gel permeation chromatography profiles, titrations with proline-rich peptides, and (15)N NMR relaxation measurements are all consistent with a monomer-dimer equilibrium with a dissociation constant on the order of 60 microM. The intermolecular interactions do, at least in part, involve proline-rich sequences in the TH region. This behavior of Btk PRR-SH3 may have implications for the functional action of Btk.