Key Residues Research Articles

Microbial rhodopsins are key light-harvesting proteins for energy conversion in aquatic environments. While typically reliant on retinal, xanthorhodopsins (XRs) also employ carotenoids as antenna pigments. Here, we investigate Kin4B8, a novel freshwater XR that exhibits a counterintuitive increase in lutein-to-retinal excitation-energy transfer (EET) efficiency under acidic conditions─from 40% at neutral pH to 55%─despite a redshift in its absorption spectrum. By integrating spectroscopic measurements with quantum chemical calculations, we examine the influence of pH-dependent protonation states of two key residues, His60 and Asp94, on EET. We find that protonation of Asp94, in particular, markedly enhances lutein-retinal electronic coupling while moderately reducing spectral overlap. The 1.24-fold increase in squared coupling compensates for the decreased overlap, resulting in a net 1.07-fold increase in the EET rate. These findings suggest that rhodopsin-carotenoid complexes employ protonation-dependent tuning of carotenoid-retinal energy transfer as a mechanism for pH-responsive adaptation to diverse light environments.

Decaprenylphosphoryl-β-D-ribose 2'-epimerase (DprE1) has emerged as one of the most promising and validated drug targets for tuberculosis (TB), owing to its essential role in the biosynthesis of arabinogalactan, a crucial component of the Mycobacterium tuberculosis (Mtb) cell wall. In the present study, a series of sixteen novel derivatives (9a-9p) were synthesized based on the structural scaffolds of the clinical trial drugs PBTZ169 and TBA7371. The synthesized compounds were characterized by NMR and LC-MS techniques. All compounds were evaluated for their antitubercular activity against the Mtb H37Rv strain. Among them, five compounds exhibited minimum inhibitory concentrations (MICs) below 25µg/mL, with compound 9m showing the most potent activity (MIC = 3.125µg/mL). Molecular docking studies revealed that compound 9m interacts with key catalytic residues His132 and Asn385 within the DprE1 binding site, and similar conformation was found upon superimposition with the standard ligand (36C). ADMET analysis demonstrated favorable pharmacokinetic and safety profiles for all synthesized derivatives. Furthermore, molecular dynamics (MD) simulations confirmed the high stability of the 9m -DprE1 complex compared to the standard reference compound. These findings suggest that compound 9m could lead to the development of novel antitubercular agents.

Background: Doxorubicin-induced cardiomyopathy is a widely used model of heart failure associated with mitochondrial dysfunction. Voltage-dependent anion channel 1 (VDAC1) oligomerization contributes to mitochondrial membrane permeabilization and cell death. Although inhibition of VDAC1 oligomerization has shown cytoprotective effects in some disease contexts, its role in doxorubicin-induced cardiac injury remains unclear, particularly in relation to mitochondrial iron homeostasis and oxidative stress. Methods: H9c2 cardiomyoblasts were treated with doxorubicin (DOX), the VDAC1 oligomerization inhibitor VBIT-4, or both. Mitochondrial respiration was assessed using the Seahorse XF analyzer. Cell viability was evaluated via propidium iodide (PI) and Hoechst staining. Mitochondrial iron levels and reactive oxygen species (ROS) were measured using Mito-FerroGreen and MitoSOX Red, respectively. To investigate the role of VDAC1 oligomerization, H9c2 cells were engineered to overexpress either wild-type VDAC1 or an oligomerization-deficient mutant (VDAC1 K53R/K274R), in which key lysine residues required for ubiquitin-mediated oligomerization were replaced with arginine. Results: VBIT-4 alone did not affect mitochondrial respiration or cell viability. However, co-treatment with DOX and VBIT-4 resulted in a significantly greater reduction in oxygen consumption rate (OCR) than DOX alone (Fig. 1A, B). PI and Hoechst staining indicated increased cell death under co-treatment.Confocal microscopy revealed that co-treatment with DOX and VBIT-4 significantly increased mitochondrial iron accumulation and ROS production compared to DOX treatment alone (Fig. 2A, B), suggesting an exacerbation of iron-driven oxidative damage. Moreover, VDAC1 K53R/K274R-overexpressing cells were more susceptible to DOX-induced mitochondrial dysfunction, showing greater reductions in basal, ATP-linked, and maximal OCRs compared to wild-type VDAC1-overexpressing cells (Fig. 3A, B), along with elevated Mito-FerroGreen and MitoSOX fluorescence. Conclusions: Unexpectedly, the inhibition of VDAC1 oligomerization exacerbated doxorubicin-induced mitochondrial dysfunction, likely through increased mitochondrial iron accumulation and oxidative stress. These findings suggest a potentially protective, context-dependent role for VDAC1 oligomerization in cardiomyocytes and call for careful consideration when targeting VDAC1 in cardiotoxic conditions.

The accumulation of foam cells, lipid-laden macrophages in atherosclerotic plaques, is a hallmark of cardiovascular disease progression. These cells contribute to chronic inflammation and plaque instability, underscoring the need for novel therapeutic strategies. Peroxisome proliferator-activated receptor gamma (PPAR-γ) is a nuclear receptor pivotal to lipid metabolism and inflammation control. Traditional Chinese Medicine (TCM) processing techniques, such as stir-frying, are believed to enhance herb efficacy, yet the molecular basis remains insufficiently understood. Here, we demonstrate that stir-fried Perilla fruit significantly increase luteolin content-a flavonoid compound capable of binding PPAR-γ at key residues (SER289, HIS323, PHE360, TYR473) as validated by molecular docking and dynamics simulation. In ox-LDL-induced RAW264.7 macrophages, luteolin promoted cholesterol efflux, reduced lipid accumulation, and upregulated PPAR-γ pathway proteins, effects that were abolished by the antagonist GW9662. These results provide mechanistic insight into the enhanced efficacy of stir-fried Perilla and highlight luteolin as a promising natural compound for atherosclerosis prevention.Graphical abstractSupplementary informationThe online version contains supplementary material available at 10.1186/s40643-025-00957-7.

To date, COVID-19 continues to pose a global health challenge, with substantial morbidity, mortality, and long-term post-COVID-19 complications threatening public health resilience. During the early pandemic, the IL-6 inhibitor (tocilizumab) was the widely used approved immunotherapy for critically ill patients; however, a subset of ICU cases exhibited normal interleukin-6 (IL-6) levels and failed to respond. We hypothesized that interleukin-17 (IL-17), which acts synergistically with IL-6, contributes to cytokine storm progression and severe inflammation. Our study uniquely integrates a clinical cross-sectional analysis with advanced in-silico modelling, directly linking patient-derived biomarker, radiological, and statistical data to molecular-level mechanisms of COVID-19 severity. Serum IL-17 was significantly elevated in critical versus moderate COVID-19 cases, with a threshold of 187.9 ng/mL predicting poor outcomes by ROC analysis. Logistic regression identified age and monocytes as independent predictors of severity, supporting a combined biomarker approach for improving the prognosis and clinical outcomes. Radiological findings, including ground-glass opacities and consolidations, alongside hematological abnormalities, were more frequent in critical cases. Computational docking revealed key amino acid residues—particularly asparagine (Asn) and cysteine (Cys)—as structural determinants shared by SARS-CoV-2 spike protein and human inflammatory mediators (IL-17R, IL-6R, CD41/CD61, CD47/SIRP). Asparaginase (ASNase) targeted critical residues such as the invariant gate residue “Asn343” and Cys213 of spike protein, Asn240 of IL-17R, and Asn136 of IL-6R. Several phytochemicals, including phytic acid and amygdalin, as well as synthetic agents such as candesartan, remdesivir, and enalapril, were found to preferentially bind to cysteine (Cys) residues—and, to a lesser extent, asparagine (Asn) residues—within key binding interfaces, in addition to targeting B-cell epitopes. This conserved residue preference supports the rationale for a dual-action therapeutic strategy in which asparaginase (ASNase) is combined with selected plant-derived ligands to simultaneously disrupt viral entry mechanisms and attenuate the inflammatory signalling. This dual-perspective approach not only identified IL-17 and IL-6 as independent severity predictors but also revealed conserved Asn and Cys motifs as critical therapeutic targets, leading to novel strategies—such as ASNase, synthetic agents and phytochemical combinations—for simultaneously blocking viral entry and modulating hyperinflammatory pathways. These findings warrant rigorous experimental and clinical validation to facilitate translation into effective therapeutic interventions.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-19359-y.

Ring contraction represents a characteristic atom-level skeletal editing strategy in natural products. While this biosynthetic approach has garnered increasing attention, its role in modifying the lactone ring size remains underexplored. Here, we report a novel δ- to γ-lactone contraction during γ-alkylidenebutenolide biosynthesis in Aspergillus candidus. This dual-enzymatic cascade involves multifunctional oxidase PesD oxidizing a PKS-derived α-pyrone to a cyclic carbonate intermediate, followed by the DUF3237 enzyme PesF excising a molecule of CO2 to achieve ring contraction─an unprecedented function for this enzyme family. Computational, crystallographic, and site-directed mutagenesis studies reveal that PesF employs a noncanonical acid-base mechanism, with Glu99 serving as a key catalytic residue that fulfills an unusual dual role.

It remains unclear whether enzymic allosteric sites on distinct secondary structure types mediate differential dynamic conformational transitions. Herein, we investigated the catalytic mechanism and secondary-structure-type-dependent allosteric regulation of β-agarase Amaga, specifically producing neoagaro-tetraose (NA4) and neoagaro-hexaose (NA6). Using homology modeling, molecular docking, point mutations, structural quantification, and kinetic analysis, we identified six key catalytic residues (Glu147, Asp149, Glu152, His169, His173, and Glu281) forming a retaining mechanism: Glu152, His169, Glu281, and Glu147 facilitate glycosylation, while Glu152 and His173 coordinate deglycosylation, with Asp149 maintaining the charge equilibrium. Notably, five β-sheet-localized residues of these six highlight their structural dominance. Allosteric site mutations revealed that random coil-localized sites enhance activity by converting random coils into α-helices, whereas β-sheet-localized sites drive transitions from β-sheets to α-helices, β-turns, and random coils. Despite differing pathways, higher-activity mutants exhibited faster structural transitions and increased NA4 production. These findings highlight secondary-structure-type-dependent design in engineering enzymes with tailored product specificity.

Discovery of novel microtubule destabilizing agents via virtual screening methods and antitumor evaluation.

Oxidases and rolling dominate black tea polysaccharide conjugates formation: Enzymatic and non-enzymatic mechanisms.

Structural basis of isethionate transport by a TRAP transporter from a sulfate-reducing bacterium.

CD44-targeted N-benzyltetrahydroisoquinoline derivatives as anticancer agents with high tumor-to-normal cell selectivity.

Identification of saltiness-enhancing compounds in Zanthoxylum bungeanum essential oil chicken soup and molecular docking with transmembrane channel-like protein 4 (TMC4).

A series of pyrrolidine-2-carbonitrile derivatives was designed, synthesized, and evaluated for their antidiabetic potential. The synthesized compounds exhibited notable inhibitory activity, with IC₅₀ values ranging from 9.36 to 21.54 µg/mL for α-amylase, 13.32 to 46.14 µg/mL for α-glucosidase, and 22.87 to 42.12 µg/mL for DPP-IV. Among the evaluated derivatives, compounds bearing para-methyl (6b) and para-chloro (6c) substituents demonstrated the most potent inhibitory activity across all three enzymatic targets. To elucidate the underlying trends, a SAR analysis was conducted, revealing that both electronic properties and steric effects of the substituents significantly influenced enzyme inhibition potency. The molecular docking studies showed strong and specific interactions between the active compounds and key residues within the catalytic sites of the target enzymes. In addition, UV-visible absorption and fluorescence spectroscopy studies demonstrated high binding affinities for both 6b and 6c with HSA, having binding constant (Ka) values of 7.31 × 10⁵ M⁻¹ and 7.43 × 10⁵ M⁻¹, respectively. Taken together, these findings highlight compounds 6b and 6c as promising lead candidates for the development of multitarget antidiabetic agents.

Identification of 1-O-Galloyl -β-D-glucose as a potent activator of Sirtuin-1: an in-silico study.

Zoonotic viruses frequently cross species barriers, but the molecular processes enabling reverse zoonosis remains poorly defined. The COVID-19 pandemic provided an unprecedented opportunity to track SARS-CoV-2 evolution in humans and its capacity to infect nonhuman primates. Our earlier analyses of primate ACE2 sequences identified key substitutions that confer resistance to wild-type (WT) SARS-CoV-2 and are conserved in New World monkeys (NWMs), establishing a strong species barrier. Using pseudovirus assays with HeLa cells expressing New World monkey ACE2 (nwmACE2), we show that while WT and Alpha strains could not infect, later variants such as Delta, Omicron, and especially the XBB lineage (XBB.1.5, EG.5.1) acquired robust infectivity toward nwmACE2. More recent lineages (JN.1, LB.1, KP.3.1.1) displayed reduced but persistent cross-species infectivity. Molecular dynamics simulations and a combined mutation prioritization framework converged key receptor-binding domain residues (including N405, N417, R452, N477, K478, A484, P486, S490, R498, and Y501) as critical contributors of adaptation to NWMs receptors. These mutations act synergistically to overcome ACE2 barriers in NWMs, correlating with documented natural infections in the field. Collectively, our findings demonstrate that human-driven viral evolution has progressively enhanced the potential of SARS-CoV-2 for cross-species transmission to nonhuman primates, underscoring the need for ongoing surveillance to mitigate future reverse zoonosis crisis.

An enhancement of fluorescence and anticancer efficiency of coumarin-substituted thiosemicarbazone and its Cu2+ therapeutic sensing.

Molecular dynamics investigation of cysteine mutations: Effects on calcium ion affinity and structural stability in the RET cysteine-rich domain.

Molecular mechanisms of Lobeline-mediated inhibition of lysozyme amyloidogenesis: A synergistic approach using biophysical and cheminformatics techniques.

Heart saver: Comprehensive investigation of (redox-) proteomic and thiol metabolite changes induced by Cana-, Dapa-, Empagliflozin treatment in 2D and 3D heart cell models reveals increased mitochondrial activity and glutathione redox defense and involvement of redox signaling.

Transglutaminase-catalyzed glycosylation of egg white peptides: Structural modulation and molecular mechanism of umami enhancement via T1R1/T1R3 interactions.