Computational Alanine Scanning Research Articles

Mechanistic Insight into the Enantioselective Degradation of Esterase QeH to (R)/(S)-Quizalofop-Ethyl with Molecular Dynamics Simulation Using a Residue-Specific Force Field.

The enantioselective mechanism of the esterase QeH against the two enantiomers of quizalofop-ethyl (QE) has been primitively studied using computational and experimental approaches. However, it is still unclear how the esterase QeH adjusts its conformation to adapt to substrate binding and promote enzyme-substrate interactions in the catalytic kinetics. The equilibrium processes of enzyme-substrate interactions and catalytic dynamics were reproduced by performing independent molecular dynamics (MD) runs on the QeH-(R)/(S)-QE complexes with a newly developed residue-specific force field (RSFF2C). Our results indicated that the benzene ring of the (R)-QE structure can simultaneously form anion-π and cation-π interactions with the side-chain group of Glu328 and Arg384 in the binding cavity of the QeH-(R)-QE complex, resulting in (R)-QE being closer to its catalytic triplet system (Ser78-Lys81-Tyr189) with the distances measured for the hydroxyl oxygen atom of the catalytic Ser78 of QeH and the carbonyl carbon atom of (R)-QE of 7.39 Å, compared to the 8.87 Å for (S)-QE, whereas the (S)-QE structure can only form an anion-π interaction with the side chain of Glu328 in the QeH-(S)-QE complex, being less close to its catalytic site. The computational alanine scanning mutation (CAS) calculations further demonstrated that the π-π stacking interaction between the indole ring of Trp351 and the benzene ring of (R)/(S)-QE contributed a lot to the binding stability of the enzyme-substrate (QeH-(R)/(S)-QE). These results facilitate the understanding of their catalytic processes and provide new theoretical guidance for the directional design of other key enzymes for the initial degradation of aryloxyphenoxypropionate (AOPP) herbicides with higher catalytic efficiencies.

Bioinformatic Analysis of Key Regulatory Genes in Adult Asthma and Prediction of Potential Drug Candidates.

Asthma is a common chronic disease that is characterized by respiratory symptoms including cough, wheeze, shortness of breath, and chest tightness. The underlying mechanisms of this disease are not fully elucidated, so more research is needed to identify better therapeutic compounds and biomarkers to improve disease outcomes. In this present study, we used bioinformatics to analyze the gene expression of adult asthma in publicly available microarray datasets to identify putative therapeutic molecules for this disease. We first compared gene expression in healthy volunteers and adult asthma patients to obtain differentially expressed genes (DEGs) for further analysis. A final gene expression signature of 49 genes, including 34 upregulated and 15 downregulated genes, was obtained. Protein-protein interaction and hub analyses showed that 10 genes, including POSTN, CPA3, CCL26, SERPINB2, CLCA1, TPSAB1, TPSB2, MUC5B, BPIFA1, and CST1, may be hub genes. Then, the L1000CDS2 search engine was used for drug repurposing studies. The top approved drug candidate predicted to reverse the asthma gene signature was lovastatin. Clustergram results showed that lovastatin may perturb MUC5B expression. Moreover, molecular docking, molecular dynamics simulation, and computational alanine scanning results supported the notion that lovastatin may interact with MUC5B via key residues such as Thr80, Thr91, Leu93, and Gln105. In summary, by analyzing gene expression signatures, hub genes, and therapeutic perturbation, we show that lovastatin is an approved drug candidate that may have potential for treating adult asthma.

PACAP key interactions with PAC1, VPAC1, and VPAC2 identified by molecular dynamics simulations

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to the glucagon/secretin family. PACAP interacts with the pituitary adenylate cyclase-activating polypeptide receptor type 1 (PAC1) and vasoactive intestinal peptide receptors 1 and 2 (VPAC1 and VPAC2), exhibiting functions in the immune, endocrine, and nervous systems. This peptide is upregulated in numerous instances of brain injury, acting as a neuroprotective agent. It can also suppress HIV-1 and SARS-CoV-2 viral replication in vitro. This work aimed to identify, in each peptide-receptor system, the most relevant residues for complex stability and interaction energy communication via Molecular Dynamics (MD), Free Energy calculations, and Protein-energy networks, thus revealing in detail the underlying mechanisms of activation of these receptors. Hydrogen bond formation, interaction energies, and computational alanine scanning between PACAP and its receptors showed that His1, Asp3, Arg12, Arg14, and Lys15 are crucial to the peptide’s stability. Furthermore, several PACAP interactions with structurally conserved positions deemed necessary in GPCR B1 activation, including Arg2.60, Lys2.67, and Glu7.42, were significant for the peptide’s stability within the receptors. According to the protein-energy network, the connection between Asp3 of PACAP and the receptors’ conserved Arg2.60 represents a critical energy communication hub in all complexes. Additionally, the ECDs of the receptors were also found to function as energy communication hubs for PACAP. Although the overall binding mode of PACAP in the three receptors was found to be highly conserved, Arg12 and Tyr13 of PACAP were more prominent in complex with PAC1, while Ser2 of PACAP was with VPAC2. The detailed analyses performed in this work pave the way for using PACAP and its receptors as therapeutic targets. Communicated by Ramaswamy H. Sarma

Assessment of Different Parameters on the Accuracy of Computational Alanine Scanning of Protein-Protein Complexes with the Molecular Mechanics/Generalized Born Surface Area Method.

Computational alanine scanning with the molecular mechanics generalized Born surface area (MM/GBSA) method constitutes a widely used approach for identifying critical residues at protein-protein interfaces. Despite its popularity, the MM/GBSA method still has certain drawbacks due to its dependence on many factors. Here, we performed a systematical study on the impact of four different parameters, namely, the internal dielectric constant, the generalized Born model, the entropic term, and the inclusion of structural waters on the accuracy of computational alanine scanning calculations with the MM/GBSA method. Our results show that the internal dielectric constant is the most critical parameter for getting accurate predictions. The introduction of entropy and interfacial water molecules decreased the quality of the predictions, while the generalized Born model had little to no effect. Considering the significance of the internal dielectric value, we proposed a methodology based on the energetic predominance of a particular set of amino acids at the protein-protein interface for selecting an appropriate value for this variable. We hope that these results serve as a guideline for future studies of protein-protein complexes using the MM/GBSA method.

Molecular Insights into Substrate Binding of the Outer Membrane Enzyme OmpT

The enzyme OmpT of the outer membrane of Escherichia coli shows proteolytic activity and cleaves peptides and proteins. Using molecular dynamics simulations in a fully hydrated lipid bilayer on a time scale of hundreds of nanoseconds, we draw a detailed atomic picture of substrate recognition in the OmpT-holo enzyme complex. Hydrogen bonds and salt bridges are essential for maintaining the integrity of the active site and play a central role for OmpT in recognizing its substrate. Electrostatic interactions are critical at all stages from approaching the substrate to docking at the active site. Computational alanine scanning based on the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) approach confirms the importance of multiple residues in the active site that form salt bridges. The substrate fluctuates along the axis of the β-barrel, which is associated with oscillations of the binding cleft formed by the residue pairs D210-H212 and D83-D85. Principal component analysis suggests that substrate and protein movements are correlated. We observe the transient presence of putative catalytic water molecules near the active site, which may be involved in the nucleophilic attack on the cleavable peptide bond of the substrate.

Molecular dynamics study on the behavior and binding mechanism of target protein Transgelin-2 with its agonist TSG12 for anti-asthma drug discovery

Transgelin-2 (TG2) is a novel promising therapeutic target for the treatment of asthma as it plays an important role in relaxing airway smooth muscles and reducing pulmonary resistance in asthma. The compound TSG12 is the only reported TG2 agonist with in vivo anti-asthma activity. However, the dynamic behavior and ligand binding sites of TG2 and its binding mechanism with TSG12 remain unclear. In this study, we performed 12.6 μs molecular dynamics (MD) simulations for apo-TG2 and TG2-TSG12 complex, respectively. The results suggested that the apo-TG2 has 4 most populated conformations, and that its binding of the agonist could expand the conformation distribution space of the protein. The simulations revealed 3 potential binding sites in 3 most populated conformations, one of which is induced by the agonist binding. Free energy decomposition uncovered 8 important residues with contributions stronger than −1 kcal/mol. Computational alanine scanning for the important residues by 100 ns conventional MD simulation for each mutated TG2-TSG12 complexes demonstrated that E27, R49 and F52 are essential residues for the agonist binding. These results should be helpful to understand the dynamic behavior of TG2 and its binding mechanism with the agonist TSG12, which could provide some structural insights into the novel mechanism for anti-asthma drug development.

Evaluation of interactions between the hepatitis C virus NS3/4A and sulfonamidobenzamide based molecules using molecular docking, molecular dynamics simulations and binding free energy calculations.

The Hepatitis C Virus (HCV) NS3/4A is an attractive target for the treatment of Hepatitis C infection. Herein, we present an investigation of HCV NS3/4A inhibitors based on a sulfonamidobenzamide scaffold. Inhibitor interactions with HCV NS3/4A were explored by molecular docking, molecular dynamics simulations, and MM/PBSA binding free energy calculations. All of the inhibitors adopt similar molecular docking poses in the catalytic site of the protease that are stabilized by hydrogen bond interactions with G137 and the catalytic S139, which are known to be important for potency and binding stability. The quantitative assessments of binding free energies from MM/PBSA correlate well with the experimental results, with a high coefficient of determination, R2 of 0.92. Binding free energy decomposition analyses elucidate the different contributions of Q41, F43, H57, R109, K136, G137, S138, S139, A156, M485, and Q526 in binding different inhibitors. The importance of these sidechain contributions was further confirmed by computational alanine scanning mutagenesis. In addition, the sidechains of K136 and S139 show crucial but distinct contributions to inhibitor binding with HCV NS3/4A. The structural basis of the potency has been elucidated, demonstrating the importance of the R155 sidechain conformation. This extensive exploration of binding energies and interactions between these compounds and HCV NS3/4A at the atomic level should benefit future antiviral drug design.

Validation of catalytic site residues of Ubiquitin Specific Protease 2 (USP2) by molecular dynamic simulation and novel kinetics assay for rational drug design.

Post-translational modifications of proteins such as protein ubiquitination are crucial for regulating conformation, stability and localization of the modified protein. Ubiquitin-specific protease 2 (USP2), a multifunctional cysteine protease is reported to be a key regulator of ubiquitylation events in numerous oncogenic proteins e.g., fatty acid synthetase, Mdm2, EGFR, cyclin A1, and cyclin-D1, etc. Thus targeting USP2 is a promising strategy for cancer therapy. USP2 is characterized by a catalytic triad comprising of cysteine, histidine and aspartic acid residues. Five residues including three from the catalytic triad and two from outside of the catalytic triad have been reported as a catalytic site of USP2 that catalyze hydrolysis and stabilizes the oxyanion formed in the intermediate step of catalysis. Here, we report two more novel residues (L269 and Y558) on USP2 involved in the catalysis of Ubiquitin using computational alanine scanning (CAS) followed by molecular dynamic simulation studies. The results obtained from CAS were further validated by a highly reliable, time- and cost-effective SDS-PAGE-based kinetics assay using UBA52 which is a natural substrate of USP2. Our results showed that mutating L269 and Y558 significantly compromised the catalytic efficiency of USP2 in hydrolyzing UBA52 which can further be extended to rational drug design of USP2 selective inhibitors and to explore the catalytic sites of other USPs. Two novel residues take part in catalytic activity of USP2 which were depicted by MD Simulations and were further validated by novel SDS-PAGE-based reliable time- and cost-effective kinetics assay.

Key Residues Affecting Binding Affinity of Sirex noctilio Fabricius Odorant-Binding Protein (SnocOBP9) to Aggregation Pheromone.

Sirex noctilio Fabricius (Hymenoptera Siricidae) is a major quarantine pest responsible for substantial economic losses in the pine industry. To achieve better pest control, (Z)-3-decen-ol was identified as the male pheromone and used as a field chemical trapping agent. However, the interactions between odorant-binding proteins (OBPs) and pheromones are poorly described. In this study, SnocOBP9 had a higher binding affinity with Z3D (Ki = 1.53 ± 0.09 μM) than other chemical ligands. Molecular dynamics simulation and binding mode analysis revealed that several nonpolar residues were the main drivers for hydrophobic interactions between SnocOBP9 and Z3D. Additionally, computational alanine scanning results indicated that five amino acids (MET54, PHE57, PHE71, PHE74, LEU116) in SnocOBP9 could potentially alter the binding affinity to Z3D. Finally, we used single-site-directed mutagenesis to substitute these five residues with alanine. These results imply that the five residues play crucial roles in the SnocOBP9-Z3D complex. Our research confirmed the function of SnocOBP9, uncovered the key residues involved in SnocOBP9-Z3D interactions, and provides an inspiration to improve the effects of pheromone agent traps.

Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering.

Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo‐activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo‐activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti‐GFP nanobodies that function robustly when directly expressed in a complex intracellular environment together with their antigen. We apply them to control subcellular protein localisation in the nematode worm Caenorhabditis elegans. Our approach applies predictions derived from computational modelling directly in a living animal and demonstrates the importance of accounting for in vivo effects on protein‐protein interactions.

A Computational Alanine Scanning of Heptapeptide RRRSAGM Targeting Dengue NS2B/NS3 Protease

The pathogenic dengue virus (DV) transmitted mainly by the Aedes aegypti mosquito has been the most rapidly spreading mosquito-borne viral disease and became a serious threat to global health issue in tropical and sub-tropical countries. The clinical symptoms ranging from mild to a fatal dengue hemorrhagic fever and dengue shock syndrome, characterized by failure of the circulation system that may lead to death. There are still no drugs or vaccines available in the market to prevent or treat dengue infections. Hence, the development of successful drugs that are safe and offer a long-lasting protection against dengue viruses is needed. Recently, the discovery of peptides with high specificity, selectivity, and efficacy; and relatively safer than small-molecule drugs have become a promising agent for a new drug. Previously, a molecular dynamic simulation reported that peptide NRRRRSAGMI from the capsid’s cleavage region had the most hydrogen bonds with NS2B/NS3 protease, thus it leads into a starting linear heptapeptide R(P3)-R(P2)-R(P1)-S(P1’)-A(P2’)-G(P3’)-M(P4’). This study aimed to investigate the importance and specificity of individual amino acid residue of the heptapeptide substrate on the binding affinity and interaction to NS2B/NS3 dengue protease through computational alanine scanning. It showed that the changing of P1 and P2 arginine to alanine resulted to higher values of binding affinity. Thus, the arginine is an important residue at the P1 and P2 positions for the binding of the substrate peptide to DENV NS2B/NS3 protease, hence the amino acid residues which are essential for the enzyme-substrate interactions can be predicted through computational alanine scanning.
 HIGHLIGHTS
 
 Dengue is a mosquito-transmitted virus cause of arthropod-borne viral disease
 The discovery of peptides became a potential agent for dengue virus and previously peptide NRRRRSAGMI has been reported as starting linear heptapeptide RRRSAGM
 A computational alanine scanning based on the peptide RRRSAGM has been performed
 S1 and S2 pockets show strong preferences for basic amino acids, whereas the S3 pocket is less selective for basic residue
 The arginine at P1 and P2 positions of the heptapeptide substrate contribute significantly to the substrate binding affinity
 
 GRAPHICAL ABSTRACT

The crystal structure of the varicella-zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity

Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, β-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein–Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.

Binding mechanism of inhibitors to SARS-CoV-2 main protease deciphered by multiple replica molecular dynamics simulations.

The outbreak caused by SARS-CoV-2 has received extensive worldwide attention. As the main protease (Mpro) in SARS-CoV-2 has no human homologues, it is feasible to reduce the possibility of targeting the host protein by accidental drugs. Thus, Mpro has been an attractive target of efficient drug design for anti-SARS-CoV-2 treatment. In this work, multiple replica molecular dynamics (MRMD) simulations, principal component analysis (PCA), free energy landscapes (FELs), and the molecular mechanics-generalized Born surface area (MM-GBSA) method were integrated together to decipher the binding mechanism of four inhibitors masitinib, O6K, FJC and GQU to Mpro. The results indicate that the binding of four inhibitors clearly affects the structural flexibility and internal dynamics of Mpro along with dihedral angle changes of key residues. The analysis of FELs unveils that the stability in the relative orientation and geometric position of inhibitors to Mpro is favorable for inhibitor binding. Residue-based free energy decomposition reveals that the inhibitor-Mpro interaction networks involving hydrogen bonding interactions and hydrophobic interactions provide significant information for the design of potent inhibitors against Mpro. The hot spot residues including H41, M49, F140, N142, G143, C145, H163, H164, M165, E166 and Q189 identified by computational alanine scanning are considered as reliable targets of clinically available inhibitors inhibiting the activities of Mpro.

Enabling biocatalysis in high-concentration organic cosolvent by enzyme gate engineering.

Biocatalysis in high-concentration organic solvents (OSs) offers many advantages, but realizing this process remains a huge challenge. An R-selective ω-amine transaminase variant (AcATAM2 ) exhibited high activity toward 50 g/L pro-sitagliptin ketone 1-[1-piperidinyl]-4-[2,4,5-trifluorophenyl]-1,3-butanedione (PTfpB). However, AcATAM2 displayed unsatisfactory organic-cosolvent resistance against high-concentration dimethyl sulfoxide (DMSO), which is required to enhance the solubility of the hydrophobic substrate PTfpB. Located in the substrate-binding tunnel, enzyme gates are structural elements that undergo reversible conformational transitions, thus affecting the accessibility of the binding pocket to solvent molecules. Depending on the conformation of the enzyme gates, one can define an open or closed conformation on which the enzyme activity in OSs may depend. To enhance the DMSO resistance of AcATAM2 , we identified the beneficial residues at the "enzyme gate" region via computational analysis, alanine scanning, and site-saturation mutagenesis. Two beneficial variants, namely, AcATAM2F56D and AcATAM2F56V , not only displayed improved enzyme activity but also exhibited enhanced DMSO resistance (the half-life value increased from 25.71 to 42.49 h under 60% DMSO). Molecular dynamic simulations revealed that the increase in DMSO resistance was mainly caused by the decrease in the number of DMSO molecules in the substrate-binding pocket. Moreover, in the kilogram-scale experiment, the conversion of 80 g/L substrate was increased from 50% (AcATAM2 ) to 85% (M2F56D in 40% DMSO) with a high e.e. of >99% within 24 h.

Molecular Dynamic Simulations of Bromodomain and Extra-Terminal Protein 4 Bonded to Potent Inhibitors.

Bromodomain and extra-terminal domain (BET) subfamily is the most studied subfamily of bromodomain-containing proteins (BCPs) family which can modulate acetylation signal transduction and produce diverse physiological functions. Thus, the BET family can be treated as an alternative strategy for targeting androgen-receptor (AR)-driven cancers. In order to explore the effect of inhibitors binding to BRD4 (the most studied member of BET family), four 150 ns molecular dynamic simulations were performed (free BRD4, Cpd4-BRD4, Cpd9-BRD4 and Cpd19-BRD4). Docking studies showed that Cpd9 and Cpd19 were located at the active pocket, as well as Cpd4. Molecular dynamics (MD) simulations indicated that only Cpd19 binding to BRD4 can induce residue Trp81-Ala89 partly become α-helix during MD simulations. MM-GBSA calculations suggested that Cpd19 had the best binding effect with BRD4 followed by Cpd4 and Cpd9. Computational alanine scanning results indicated that mutations in Phe83 made the greatest effects in Cpd9-BRD4 and Cpd19-BRD4 complexes, showing that Phe83 may play crucial roles in Cpd9 and Cpd19 binding to BRD4. Our results can provide some useful clues for further BCPs family search.

The eptinezumab:CGRP complex structure – the role of conformational changes in binding stabilization

ABSTRACT To further elucidate the mechanism of action and binding properties of eptinezumab to calcitonin gene-related peptide (CGRP), X-ray crystallography, computational alanine scanning, and molecular dynamics were used. X-ray diffraction data were collected to determine the three-dimensional structures of the unbound eptinezumab antigen-binding fragment (Fab) and the Fab:CGRP complex. Molecular dynamics simulations were performed to analyze the transition between uncomplexed and complex states. The amidated C-terminus of CGRP was shown to bind in a pocket formed by the Fab heavy and light chains. There was extensive contact between all six complementarity-determining regions (CDRs; composed of light-chain [L1, L2, and L3] and heavy-chain [H1, H2, H3]) of eptinezumab and CGRP. The complex demonstrated a high ligand-binding surface area dominated by aromatic residues. CDR L3 contains a disulfide bond that stabilizes the loop, contributes surface area to the binding pocket, and provides van der Waals contacts. Comparison of the uncomplexed and complex structures revealed motion near the binding cleft. The CDR loops H2 and H3 were displaced ~1.4–2.0 Å and residue H-Tyr33 changed conformation, creating a ‘latch-and-lock’ mechanism for binding CGRP and preventing dissociation. Computational alanine scanning of CGRP identified energetic ‘hot spots’ that contribute to binding energy; mutating these positions to residues in homologous neuropeptides resulted in unfavorable binding energies. The attributes of the Fab region and the conformational changes that occur in eptinezumab during binding to CGRP contribute to the specificity, durability, and strength of the interaction, and likely underlie the rapid and sustained migraine preventive effect observed in clinical studies.

Computational Alanine Scanning Reveals Common Features of TCR/pMHC Recognition in HLA-DQ8-Associated Celiac Disease.

In HLA-DQ8-associated celiac disease, Gliadin-γ1 or Gliadin-α1 peptide is presented to the cell surface and recognized by several types of T-cell receptor (TCR), but it is still unclear how the TCR, peptide, and the major histocompatibility complex (MHC) act together to trigger celiac disease. For now, most of the analysis is based on static crystal structures. And the detailed information about these structures based on energetic interaction is still lacking. Here, we took four types of celiac disease-related MHC-peptide-TCR structures from three patients to perform computational alanine scanning calculations using the molecular mechanics generalized born surface area (MM/GBSA) approach combined with a recently developed interaction entropy (IE) method to identify the key residues on TCR, peptide, and MHC. Our study aims to shed some light on the interaction mechanism of this complex protein interaction system. Based on detailed computational analysis and mutational calculations, important binding interactions in these triple-interaction complexes are analyzed, and critical residues responsible for TCR/pMHC recognition pattern in HLA-DQ8-associated celiac disease are presented. These detailed analysis and computational result should help shed light on our understanding of the celiac disease and the development of the medical treatment.

Computational Screening for the Anticancer Potential of Seed-Derived Antioxidant Peptides: A Cheminformatic Approach.

Some seed-derived antioxidant peptides are known to regulate cellular modulators of ROS production, including those proposed to be promising targets of anticancer therapy. Nevertheless, research in this direction is relatively slow owing to the inevitable time-consuming nature of wet-lab experimentations. To help expedite such explorations, we performed structure-based virtual screening on seed-derived antioxidant peptides in the literature for anticancer potential. The ability of the peptides to interact with myeloperoxidase, xanthine oxidase, Keap1, and p47phox was examined. We generated a virtual library of 677 peptides based on a database and literature search. Screening for anticancer potential, non-toxicity, non-allergenicity, non-hemolyticity narrowed down the collection to five candidates. Molecular docking found LYSPH as the most promising in targeting myeloperoxidase, xanthine oxidase, and Keap1, whereas PSYLNTPLL was the best candidate to bind stably to key residues in p47phox. Stability of the four peptide-target complexes was supported by molecular dynamics simulation. LYSPH and PSYLNTPLL were predicted to have cell- and blood-brain barrier penetrating potential, although intolerant to gastrointestinal digestion. Computational alanine scanning found tyrosine residues in both peptides as crucial to stable binding to the targets. Overall, LYSPH and PSYLNTPLL are two potential anticancer peptides that deserve deeper exploration in future.

Computational Interaction Analysis of Sirex noctilio Odorant-Binding Protein (SnocOBP7) Combined with Female Sex Pheromones and Symbiotic Fungal Volatiles

Sirex noctilio, a major forestry quarantine pest, has spread rapidly and caused serious harm. However, existing methods still need to be improved because its olfactory interaction mechanisms are poorly understood. In order to study the role of male-specific protein SnocOBP7 in the protein–ligand interactions, we selected it as the object of computational simulation and analysis. By docking it with 11 ligands and evaluating free binding energy decomposition, the three best binding ligands were found to be female sex pheromones ((Z)-7-heptacosene and (Z)-7-nonacosene) and symbiotic fungal volatiles ((−)-globulol). Binding mode analysis and computational alanine scanning suggested that five residues play key roles in the binding of each female sex pheromone to SnocOBP7, whereas two residues play key roles in (−)-globulol binding. Phe108 and Leu36 may be the crucial sites via which SnocOBP7 binds female sex pheromones, whereas Met40 may regulate the courtship behavior of males, and Leu61 may be related to mating and host finding. Our studies predicted the function of SnocOBP7 and found that the interaction between SnocOBP7 and pheromone is a complex process, and we successfully predicted its binding key amino-acid sites, providing a basis for the development of new prevention and control methods relying on female sex pheromones and symbiotic fungi.

Identification of Hotspot Residues in Binding of SARS-CoV-2 Spike and Human ACE2 Proteins

Coronaviruses are a large family of viruses that can cause respiratory infections with varying severity from common cold to severe diseases such as novel coronavirus disease (COVID-19). COVID-19 has been declared as a global pandemic by the World Health Organization on March 11, 2020 and with the development of vaccines it slowed down as of this date. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses its spike glycoprotein (Sgp) to bind human angiotensin-converting enzyme 2 (hACE2) receptor, and mediates membrane fusion and virus entry. The recognition of Sgp to human ACE2 and its high affinity for it has been of great importance since this provides the first step in viral entry to human cells. Therefore, it is crucial to identify key residues (hotspots) in this process. In this study, computational alanine scanning has been performed for Sgp and hACE2. The residues identified with significance in binding and other residues in close proximity were studied further through molecular mechanics-based protein binding free energy change prediction methods. Moreover, the interfacial residues in both proteins were investigated for their cooperative binding. Additionally, folding free energy changes upon mutation to Ala were calculated to assess their effect on stability of Sgp and hACE2. Our results taken together with findings from previous studies revealed the residues that are most significant and are relatively significant in binding of Sgp to human ACE2 protein.