Search Results (254)

Background/Objectives: Understanding the molecular interactions between small bioactive compounds and serum albumins is essential for drug development and pharmacokinetics. Noraucuparin, a biphenyl-type phytoalexin with promising pharmacological properties, has shown a strong binding affinity to bovine serum albumin (BSA), a model protein for drug transport. This study aims to elucidate the structural and energetic characteristics of the noraucuparin–BSA complex under physiological and slightly elevated temperatures. Methods: Microsecond-scale molecular dynamics (MD) simulations and Molecular Mechanics Generalized Born Surface Area (MMGBSA)-binding-free energy calculations were performed to investigate the interaction between noraucuparin and BSA at 298 K and 310 K. Conformational flexibility and per-residue energy decomposition analyses were conducted, along with interaction network mapping to assess ligand-induced rearrangements. Results: Noraucuparin preferentially binds to site II of BSA, near the ibuprofen-binding pocket, with stabilization driven by hydrogen bonding and hydrophobic interactions. Binding at 298 K notably increased the structural mobility of BSA, affecting its global conformational dynamics. Key residues, such as Trp213, Arg217, and Leu237, contributed significantly to complex stability, and the ligand induced localized rearrangements in the protein’s intramolecular interaction network. Conclusions: These findings offer insights into the dynamic behavior of the noraucuparin–BSA complex and enhance the understanding of serum albumin–ligand interactions, with potential implications for drug delivery systems. Full article

The relationship between protein structure and function is intrinsically interconnected, as the structure of a protein directly determines its functional properties. To investigate the effects of temperature and pressure on protein function, this study employed ethyl carbamate (EC) hydrolase as a model food enzyme and conducted molecular dynamics (MD) simulations under varying temperature and pressure levels to elucidate its structure–function relationship. By systematically analyzing the dynamic changes in root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), hydrogen bonding, catalytic pocket conformation, and packing density under different temperature and pressure conditions, we revealed the structural adaptability of EC hydrolase. Furthermore, we analyzed the characteristics of EC hydrolase using molecular dynamics simulations with temperature and pressure levels, as well as conformational bias-based computer-aided engineering, providing both theoretical and experimental foundation for the adaptability mechanisms of enzymes under extreme conditions. Full article

Background/Objectives: Chloroquine (CQ) and hydroxychloroquine (HCQ) have been the subject of debate in the treatment of COVID-19 due to the lack of conclusive evidence regarding their efficacy and safety. Our study aims to investigate the molecular interaction between the enantiomers of CQ and HCQ with angiotensin-converting enzyme 2 (ACE2), focusing on the binding mechanism, affinity, and selectivity. Methods: We used in silico methods, including molecular docking, molecular dynamics, and binding free energy calculations using the MM-PBSA method, to evaluate the interaction between the enantiomers of CQ and HCQ with ACE2. Results: We identified three main interaction sites on ACE2 (α, β, and γ) with distinct characteristics based on the pocket size, hydrophilic/hydrophobic characteristics, and affinity energy. We observed that protonation states and ionic strength significantly influence the binding affinity and specificity. In particular, the selectivity of the β-site, characterized by its smaller size and hydrophilic residues, is preferential for species with the (R) configuration, whereas the α and γ binding sites, with a larger size and amphiphilic residues, have greater affinity for the (S) enantiomer of CQ and HCQ. Furthermore, ionic strength can affect ligand binding by modulating electrostatic interactions, molecular conformation, solvation, and the stability of the complex. Conclusions: Our findings reveal that protonation states and the ionic strength substantially impact the binding affinity and specificity, regulated by spatial and polar–electrostatic complementarity, as well as hydrophobic contributions. These results suggest that understanding the interaction between CQ and HCQ enantiomers with ACE2 could be useful for the design of novel therapies against COVID-19. Full article

Riboswitches regulate gene expression through intricate dynamic conformational transitions, with divalent cation Mg²⁺ and their ligands playing pivotal roles in this process. The dynamic structural mechanism by which the S-adenosyl-L-methionine (SAM) responsive SAM-VI riboswitch (riboSAM) regulates the downstream SAM synthase gene translation remains unclear. In this study, we employed position-selective labeling of RNA (PLOR) to incorporate Cy3-Cy5 into designated positions of riboSAM, applying single-molecule Förster resonance energy transfer (smFRET) method to track its conformational switches in response to Mg²⁺ and SAM. smFRET analysis revealed that in the absence of Mg²⁺ and ligand, riboSAM predominantly adopted a translation-activating apo conformation. Physiological concentrations of Mg²⁺ induced riboSAM to fold into dynamic transit-p and holo-p states, creating a transient and structurally pliable binding pocket for ligand binding. SAM binding locks the dynamic transit-p and holo-p states into their final stable transit and holo conformations through conformational selection, turning off downstream cis-gene expression and completing feedback regulation of cellular SAM concentration. The observed synergistic regulatory effect of Mg²⁺ ions and ligand on riboSAM’s conformational dynamics at single-molecule resolution provides new mechanistic insights into gene regulation by diverse riboswitch classes. Full article

KRAS is a pivotal oncoprotein that regulates cell proliferation and survival through interactions with downstream effectors such as RAF1. Despite significant advances in understanding KRAS biology, the structural and dynamic mechanisms of KRAS allostery remain poorly understood. In this study, we employ microsecond molecular dynamics simulations, mutational scanning, and binding free energy calculations together with dynamic network modeling to dissect how engineered DARPin proteins K27, K55, K13, and K19 engage KRAS through diverse molecular mechanisms ranging from effector mimicry to conformational restriction and allosteric modulation. Mutational scanning across all four DARPin systems identifies a core set of evolutionarily constrained residues that function as universal hotspots in KRAS recognition. KRAS residues I36, Y40, M67, and H95 consistently emerge as critical contributors to binding stability. Binding free energy computations show that, despite similar binding modes, K27 relies heavily on electrostatic contributions from major binding hotspots while K55 exploits a dense hydrophobic cluster enhancing its effector-mimetic signature. The allosteric binders K13 and K19, by contrast, stabilize a KRAS-specific pocket in the α3–loop–α4 motif, introducing new hinges and bottlenecks that rewire the communication architecture of KRAS without full immobilization. Network-based analysis reveals a strikingly consistent theme: despite their distinct mechanisms of recognition, all systems engage a unifying allosteric architecture that spans multiple functional motifs. This architecture is not only preserved across complexes but also mirrors the intrinsic communication framework of KRAS itself, where specific residues function as central hubs transmitting conformational changes across the protein. By integrating dynamic profiling, energetic mapping, and network modeling, our study provides a multi-scale mechanistic roadmap for targeting KRAS, revealing how engineered proteins can exploit both conserved motifs and isoform-specific features to enable precision modulation of KRAS signaling in oncogenic contexts. Full article

Protein glycosylation plays a crucial role in cancer biology, influencing essential cellular processes such as cell signalling, immune recognition, and tumour metastasis. Therefore, this study highlights the therapeutic potential of targeting glycosylation in cancer treatment, as modulating these modifications could disrupt the fundamental mechanisms driving cancer progression and improve therapeutic outcomes. Recently, Tunicamycin C, a well-known glycosylation inhibitor, has shown promise in breast cancer treatment but remains unexplored in colorectal cancer (CRC). Thus, in this study, we aimed to understand the potential action of Tunicamycin C in CRC using in silico studies to identify possible drug targets for Tunicamycin C. First, we identified two target proteins using the HTDocking algorithm followed by GO and KEGG pathway searches: thymidine kinase 1 (TK1) and cAMP-dependent protein kinase catalytic subunit alpha (PKAc). Following this, molecular dynamics modelling revealed that Tunicamycin C binding induced a conformational perturbation in the 3D structures of TK1 and PKAc, inhibiting their activities. This interaction led to a stable design, promoting optimal binding of Tunicamycin C in the hydrophobic pockets of TK1 and PKAc. Serial validation studies highlighted the role of active site residues in binding stabilisation. Tunicamycin C exhibited high binding affinity with TK1 and PKAc. This study provides a way to explore and repurpose novel inhibitors of TK1 and PKAc and identify new therapeutic targets, which may block glycosylation in cancer treatment. Full article

Comparative studies using lysophosphatidic acid (LPA) and the synthetic agonist, oleoyl-methoxy glycerophosphothionate (OMPT), in cells expressing the LPA₃ receptor revealed differences in the action of these agents. The possibility that more than one recognition cavity might exist for these ligands in the LPA₃ receptor was considered. We performed agonist docking studies exploring the whole protein to obtain tridimensional details of the ligand–receptor interaction. Functional in cellulo experiments using mutants were also executed. Our work includes blind docking using the unrefined and refined proteins subjected to hot spot predictions. Distinct ligand protonation (charge −1 and −2) states were evaluated. One LPA recognition cavity is located near the lower surface of the receptor close to the cytoplasm (Lower Cavity). OMPT displayed an affinity for an additional identification cavity detected in the transmembrane and extracellular regions (Upper Cavity). Docking targeted to Trp102 favored binding of both ligands in the transmembrane domain near the extracellular areas (Upper Cavity), but the associating amino acids were not identical due to close sub-cavities. A receptor model was generated using AlphaFold3, which properly identified the transmembrane regions of the sequence and co-modeled the lipid environment accordingly. These two models independently generated (with and without the membrane) and adopted essentially the same conformation, validating the data obtained. A DeepSite analysis of the model predicted two main binding pockets, providing additional confidence in the predicted ligand-binding regions and support for the relevance of the docking-based interaction models. In addition, mutagenesis was performed of the amino acids of the two detected cavities. In the in cellulo studies, LPA action was much less affected by the distinct mutations than that of OMPT (which was almost abolished). Therefore, docking and functional data indicate the presence of distinct agonist binding cavities in the LPA₃ receptor. Full article

Diaporthe species are critical plant pathogens that contribute to a disease complex responsible for substantial yield losses in soybean production worldwide. However, reports on the primary Diaporthe species causing soybean stem blight and their sensitivity to various fungicides are scarce in China. In this study, a total of 46 D. longicolla strains were isolated and identified from diseased soybean stems and rots collected from 14 regions of Heilongjiang province in 2021 and 2022. Among the eight fungicides examined, fludioxonil, mefentrifluconazole, tebuconazole, and azoxystrobin demonstrated effective inhibition for D. longicolla, with EC₅₀ values < 0.3 µg/mL. Interestingly, the EC₅₀ values of D. longicolla to two succinate dehydrogenase inhibitors (SDHIs), pydiflumetofen and fluopyram, were 5.47 µg/mL and over 100 µg/mL, respectively. In molecular dynamics simulations, pydiflumetofen exhibited a smaller RMSD, while fluopyram had a higher binding free energy with Sdh proteins compared to pydiflumetofen. This difference may contribute to the higher activity of pydiflumetofen in D. longicolla. Further analysis of the electrostatic potential and structural conformations of the binding pocket revealed that pydiflumetofen formed more hydrophobic interactions with SdhC and SdhD and was positioned closer to the SdhD subunit. A mixture of fludioxonil and mefentrifluconazole at a ratio of 1:5, as well as fludioxonil and pydiflumetofen at a ratio of 1:5, exhibited synergistic effects. These findings demonstrated that several fungicides could be utilized to control Diaporthe stem blight, and the difference in binding affinity to the Sdh subunit impacts sensitivity to fluopyram and pydiflumetofen. Full article

Inflammation contributes to the onset and development of many diseases, including neurodegenerative diseases, caused by the activation of microglia, leading to neurological deterioration. Nuclear factor-κB (NF-κB) is one of the most relevant pathways for identifying anti-inflammatory molecules. In this study, polygodial and isotadeonal, two drimane sesquiterpene dialdehydes, were isolated from Drimys winteri, a medicinal tree of the Mapuche people in Chile. Isotadeonal, or epi-polygodial, was obtained from polygodial by epimerization in basic media (60% yield, Na₂CO₃, r/t, 24 h). Both sesquiterpenoids were evaluated on the NF-κB pathway, with the result that isotadeonal inhibited the phosphorylation of IκB-α at 10 μM with higher potency by Western blotting. The final inhibition of the pathway was evaluated using a SEAP reporter (secreted alkaline phosphatase) on THP-1 cells. Isotadeonal inhibited SEAP with higher potency than polygodial, quercetin, and CAPE (phenethyl ester of caffeic acid). In silico analysis suggests that the α-aldehyde of isotadeonal adopts a more stable conformation in the active pocket of IκB-α than polygodial. Full article

Background: The four subtypes of G protein-coupled receptors (GPCRs) regulated by histamine play critical roles in various physiological and pathological processes, such as allergy, gastric acid secretion, cognitive and sleep disorders, and inflammation. Previous experimental structures of histamine receptors (HRs) with agonists and antagonists exhibited multiple conformations for the ligands and G protein binding. However, the structural basis for HR regulation and signaling remains elusive. Methods: We determined the cryo-electron microscopy (cryo-EM) structure of the H4R-histamine-Gi complex at 2.9 Å resolution, and predicted the models for all four HRs in the ligand-free apo and G protein subtype binding states using AlphaFold3 (AF3). Results: By comparing our H4R structure with the experimental HR structures and the computational AF3 models, we elucidated the distinct histamine binding modes and G protein interfaces, and proposed the essential roles of Y^6.51 and Q^7.42 in receptor activation and the intracellular loop 2 (ICL2) in G protein bias. Conclusions: Our findings deciphered the molecular mechanisms underlying the regulation of different HRs, from the extracellular ligand-binding pockets and transmembrane motifs to the intracellular G protein coupling interfaces. These insights are expected to facilitate selective drug discovery targeting HRs for diverse therapeutic purposes. Full article

2′-Deoxythymidine-5′-monophosphate, dTMP, is an essential precursor of thymine, one of the four canonical bases of DNA. In almost all living organisms, dTMP is synthesized de novo by a reductive methylation reaction of 2′-deoxyuridine-5′-monophosphate (dUMP) catalyzed by the thymidylate synthase, where the carbon used for the methylation is derived from methylenetetrahydrofolate (CH2THF). Many microbes, including human pathogens, utilize the flavin-dependent thymidylate synthase encoded by the thyX gene to generate dTMP. The mechanism of action relies on the reduced coenzyme FADH⁻, which acts both as a mediator, facilitating methylene transfer from CH2THF to dUMP, and as a reducing agent. Here, we present for the first-time crystallographic structures of ThyX from Thermotoga maritima in the reduced state alone and in complex with dUMP. ThyX flavin reduction appears to order the active site, favoring a flavin conformation that drastically deviates from that observed in the oxidized enzyme. The structures show that FADH⁻ potentially controls access to the folate site and the conformation of two active site loops, affecting the degree of accessibility of substrate pockets to the solvent. Our results provide the molecular basis for the sequential enzyme mechanism implemented by ThyX during dTMP biosynthesis. Full article

The regioselective glycosylation and product specificity of hydroxyflavonoid compounds profoundly influences their biological activity and stability, offering significant therapeutic potential. However, most cyclodextrin glucosyltransferases (CGTases) inherently lack regioselectivity and product specificity for flavone glycosylation. Herein, a CGTase from Paenibacillus macerans was engineered for enhanced glycosylation regioselectivity and product specificity by combining molecular docking analysis and saturation mutagenesis strategies. K232L (favoring 4′-and 6-hydroxyflavones) and K232V (favoring 7-hydroxyflavone) were identified with distinct preferences. In addition, H233Y (preferring for 4′-hydroxyflavones), H233T (preferring for 6′-hydroxyflavones), and H233K (preferring for 7′-hydroxyflavones) also demonstrated distinct regioselectivity. These variants further exhibited enhanced hydrolytic activity, enabling the efficient production of short sugar-chain glycosides. Molecular dynamics (MDs) simulations revealed that the variants adopted optimized catalytic conformations with increased loop region flexibility near the binding pocket, enhancing substrate accessibility. These findings underscore the pivotal roles of K232 and H233 in broadening the substrate scope of CGTase and offer valuable guidance for enzyme engineering targeting regioselective glycosylation. Full article

The hydroxylated perhydroquinoxaline 14 was designed by conformational restriction of the prototypical κ receptor agonist U-50,488 and the introduction of an additional polar group. The synthesis of 14 comprised ten reaction steps starting from diethyl 3-hydroxyglutarate (4). The first key step was the diastereoselective establishment of the tetrasubstituted cyclohexane 7 by the reaction of dialdehyde 6 with benzylamine and nitromethane. The piperazine ring was annulated by the reaction of silyloxy-substituted cyclohexanetriamine 8 with dimethyl oxalate. The pharmacophoric structural elements characteristic for κ receptor agonists were finally introduced by functional group modifications. The structure including the relative configuration of the tetrasubstituted cyclohexane derivative (2r,5s)-7a and the perhydroquinoxaline 9 was determined unequivocally by X-ray crystal structure analysis. The hydroxylated perhydroquinoxaline 14 showed moderate κ receptor affinity (K_i = 599 nM) and high selectivity over μ, δ, σ₁, and σ₂ receptors. An ionic interaction between the protonated pyrrolidine of 14 and D138 of κ receptor anchors 14 in the κ receptor binding pocket. Full article

P21-activated kinase 4 (PAK4) plays a crucial role in the proliferation and metastasis of various cancers. However, developing selective PAK4 inhibitors remains challenging due to the high homology within the PAK family. Therefore, developing highly selective PAK4 inhibitors is critical to overcoming the limitations of existing inhibitors. We analyzed the structural differences in the binding pockets of PAK1 and PAK4 by combining cross-docking and molecular dynamics simulations to identify key binding regions and unique structural features of PAK4. We then performed screening using shape and protein conformation ensembles, followed by a re-evaluation of the docking results with deep-learning-driven GNINA to identify the candidate molecule, STOCK7S-56165. Based on this, we applied a fragment-replacement strategy under electrostatic-surface-matching conditions to obtain Compd 26. This optimization significantly improved electrostatic interactions and reduced binding energy, highlighting its potential for selectivity. Our findings provide a novel approach for developing selective PAK4 inhibitors and lay the theoretical foundation for future anticancer drug design. Full article

G protein-coupled receptors (GPCRs) play essential roles in numerous physiological processes and are key targets for drug development. Among them, adhesion GPCRs (aGPCRs) stand out for their unique domain structures and diverse functions. ADGRG2 is a member of the aGPCR family and is involved in the regulation of various systems in the human body, including reproductive, nervous, cardiovascular, and endocrine systems. Investigating ADGRG2 antagonists enhances our understanding of its regulatory roles in diverse physiological processes, yet their precise mechanisms of action remain unclear. To address this, we investigated the antagonistic mechanism of ADGRG2 by examining its interactions with various antagonists, including short peptides (F601D, F601E) and small molecules (deoxycorticosterone, DOC). Using advanced metadynamics simulation, ligand binding assay and cAMP assay, we elucidated the binding modes of these antagonists. We identified five distinct F601D-ADGRG2 complex states, four F601E-ADGRG2 complex states, and three DOC-ADGRG2 complex states, which were each characterized by specific hydrogen bonds or polar interactions with their respective ligands. Although the ADGRG2 binding pocket consists of both polar and hydrophobic residues, our biochemical experiments revealed that mutations in polar amino acids significantly reduce the efficacy of the antagonists. Our results show that F601D, F601E, and DOC induce the formation of Y758^ECL2-N775^5.32-N860^7.46 polar networks within ADGRG2, effectively stabilizing its inactive state. Additionally, we compared the active and inactive states of ADGRG2, highlighting the structural changes induced by antagonist-stabilized polar networks and their impact on receptor conformation. These findings provide important insights into the biology of aGPCRs and provide theoretical support for the rational design of therapeutic drugs targeting ADGRG2. Full article