Proteins[JOURNAL]
Sun
200 papers
Correction to Structural Classification Insights Into the Plant Defensive Peptides.
Proteins · 2026 Feb · PMID 41735235 · Publisher ↗
Abstract loading — click title to view on PubMed.
In Silico and In Vitro Analysis of Ganoderic Acid A Binding to Human Monocarboxylate Transporters 1 and 4.
Bashir MA, Shao CS, Abdalla M … +3 more , Yu X, Fetisoa MR, Huang Q
Proteins · 2026 Aug · PMID 41735189 · Publisher ↗
In recent years, the search for novel anticancer agents has increasingly focused on monocarboxylate transporters (MCTs) because of their involvement in tumor metabolism. Ganoderic acid A (GAA), a triterpenoid derived fro...
In recent years, the search for novel anticancer agents has increasingly focused on monocarboxylate transporters (MCTs) because of their involvement in tumor metabolism. Ganoderic acid A (GAA), a triterpenoid derived from the medicinal mushroom Ganoderma lucidum, has demonstrated anticancer potential; however, its interaction with MCT isoforms remains insufficiently characterized. In this study, we examined the interaction between GAA and MCT1 and MCT4 using complementary computational and experimental approaches. Full-length structures of MCT1 and MCT4 were predicted using AlphaFold2 and validated with the SAVES server. Molecular docking and molecular dynamics simulations using the known dual inhibitor syrosingopine as a reference indicated that GAA can associate with both MCT1 and MCT4. Cellular thermal shift assay (CETSA) and isothermal dose-response fingerprinting (ITDRF) showed that GAA thermally destabilized both MCT1 and MCT4, supporting a direct protein-compound interaction. Notably, ITDRF analysis revealed enhanced stability of a higher-molecular-weight MCT4, suggesting a biphasic binding behavior. Together, these findings indicate that GAA directly interacts with MCT1 and MCT4, and uncovers a biphasic binding pattern associated with MCT4.
Bioinformatics-Driven Design and Evaluation of Recombinant Multi-Epitope Immunogens Derived From Snake Venom Toxins as Potential Antivenom Candidates.
Maoz H, Elalouf A
Proteins · 2026 Jul · PMID 41730794 · Publisher ↗
Snakebite envenomation is a major public health concern, particularly in low- and middle-income regions where access to safe and effective antivenoms is limited. Traditional antivenoms, derived from immunization with cru...
Snakebite envenomation is a major public health concern, particularly in low- and middle-income regions where access to safe and effective antivenoms is limited. Traditional antivenoms, derived from immunization with crude venom, often trigger adverse reactions and lack specificity against key venom components. This study presents a bioinformatics-driven approach to design, construct, and evaluate a novel panel of recombinant multi-epitope toxin-derived immunogens targeting the most clinically significant snake venom toxin families. Five principal toxin families-three-finger toxins (3FT), phospholipase A2 (PLA), snake venom metalloproteinases (SVMP), snake venom serine proteases (SVSP), and dendrotoxins (DDT)-were computationally analyzed to identify conserved, antigenic, non-toxic, and non-allergenic B-cell and T-cell epitopes. Multi-epitope recombinant constructs were designed using linker systems, a TLR4 agonist adjuvant, and an MITD-signal sequence to enhance immunogenicity. Structural modeling, refinement, and validation were performed using AlphaFold 3 and GalaxyRefine. Protein-TLR interactions were assessed using molecular docking with ClusPro, selecting the top-ranked pose based on the lowest energy score for initial analysis, as these often correspond to the most stable configurations. Normal mode analysis (NMA), molecular dynamics simulation (MDS), and post-simulation analysis were used to evaluate the stability of the complexes. While in silico immune simulations evaluated the immunogenic potential of the constructs. The designed multi-epitope toxin-derived immunogens were predicted to have favorable physicochemical properties, including molecular weights ranging from 33.7 to 56.37 kDa, basic isoelectric points, high thermostability (aliphatic index: 66.72-77.08), and hydrophilicity (negative GRAVY scores). Refined 3D models exhibited more than 93% residues in Ramachandran favored regions, suggesting structural reliability. Molecular docking revealed strong and stable interactions with TLR2 and TLR4, particularly in the SVMP-TLR4 complex (ΔG = -21.0 kcal/mol; K = 3.7 × 10 M). NMA, MDS, and post-simulation analysis collectively showed that 3FT immunogen complexes with TLR2/4 were the most stable and compact with coordinated motions, PLA and DDT displayed moderate flexibility with maintained integrity, SVSP showed intermediate instability, and SVMP, particularly with TLR2, exhibited pronounced conformational instability and dynamic disorder, highlighting clear receptor- and toxin-dependent differences in stability and collective behavior. Immune simulations theoretically predicted robust humoral and cellular immune responses, with early IgM/IgG production, expansion of B-cell and T-cell populations, and balanced cytokine profiles indicative of a safe immunogenic response. This study provides in silico evidence suggesting the potential of recombinant multi-epitope toxin-derived immunogens as a next-generation therapeutic strategy for snakebite management. The designed constructs may offer improvements in specificity, safety, and manufacturability over traditional antivenoms, providing a promising foundation for further experimental validation and clinical translation. Future refinements could incorporate cluster overlap evaluation to mitigate bias from single-pose selection, particularly for poses with overlapping scores.
Comparative Binding Dynamics of Minibinder 8.6 and HBD3 With TLR3 as Adjuvants for Developing a Peptide-Based Multi-Epitope Subunit Vaccine Against mCRPC: A Molecular Dynamics Study.
Paul I, Ray S, Tewari M … +2 more , Saha M, Ray S
Proteins · 2026 Jul · PMID 41720627 · Publisher ↗
Metastatic castration resistance prostate cancer (mCRPC) is the advanced state of prostate cancer where majority of patients succumb to ineffective treatment perspectives like androgen deprivation alongside salvage thera...
Metastatic castration resistance prostate cancer (mCRPC) is the advanced state of prostate cancer where majority of patients succumb to ineffective treatment perspectives like androgen deprivation alongside salvage therapies. mCRPC is predominantly orchestrated by androgen receptor (AR)-dependent gene expression. On the account of AR being a "potentially attractive immunological target", an immunoinformatics pipeline was built to identify and screen mutation-independent, broad MHC covering, potential antigenic, non-allergenic, non-toxic and soluble epitopes. The filtered epitopes required assembly into a unified construct with interconnecting linkers and adjuvant and further TLR3-docking. We chose TLR3 because of its pro-apoptotic activity in prostate cancer, to check active immune response by the vaccine. Our study was not confined to the use of a conventional adjuvant like human beta defensin-3 but it extended to the scope of utilization of a protein-based TLR3-specific agonist as an adjuvant for assembling the second composite construct. Our path was guided by the discovery of TLR3-specific agonist minibinder 8.6 (a small, hyperstable protein) by Adams et al. An extensively comparative molecular dynamics study of the free state and bound states of the two constructs unveiled a more stable interaction, complex stability and immune response attributing to the specificity of the minibinder-based construct towards TLR3. Our work circumscribes a multi-headed approach beginning with peptide subunit multi-epitope vaccine construct design for mitigating mCRPC; secondarily, endorsing the advocacy of TLR3-agonizing minibinders as vaccine adjuvants for enhanced immunity and finally posing a comparative framework of minibinder 8.6 over HBD3, as a more potential adjuvant, apprehending wet-lab proof.
AlphaFold2-Guided Cyclic Peptide Stabilizer Design to Target Protein-Protein Interactions.
Halbwedl N, Zacharias M
Proteins · 2026 Jul · PMID 41696819 · Publisher ↗
The control and modulation of protein-protein interactions (PPIs) is of central importance for the majority of biological processes and most biomedical applications. Stabilization of PPIs, besides inhibition, is of growi...
The control and modulation of protein-protein interactions (PPIs) is of central importance for the majority of biological processes and most biomedical applications. Stabilization of PPIs, besides inhibition, is of growing pharmaceutical interest. Due to their small size, drug-like organic molecules may not provide sufficient interaction surfaces to allow for high-affinity dual binding to both partners of a protein-protein complex. Cyclic peptides offer larger interaction surfaces, making them a promising class of stabilizers of PPIs. We have developed a computational protocol to rapidly and systematically design cyclic peptides that optimize not only the interaction with one target protein but simultaneously optimize the dual binding to two protein partners. The cyclic peptide generation is based on a modified AlphaFold2-based peptide design approach and combines confidence scoring with force field-based scoring using Molecular Dynamics simulations. The performance of the method is tested on protein-protein complexes with known cyclic peptide binders and stabilizers. In addition, the approach is used to design cyclic peptides that can act as bifunctional molecules, recruiting the cellular protein degradation system to a target protein. The designed cyclic peptides achieve similar or better calculated interaction scores than known binders and exhibit well-balanced interactions with both protein partners. The design protocol is generally applicable to cyclic peptide design for modulating or inducing protein-protein association and could be useful for many biomedical design efforts.
Computationally Efficient Network Models Successfully Predict Allosteric Sites of SARS-CoV-2 Main Protease and Reveal Its Dynamic Allostery.
Yuce M, Kurkcuoglu O
Proteins · 2026 Jul · PMID 41689234 · Publisher ↗
Developing allosteric drugs to treat pathogenic diseases can offer a promising alternative to orthosteric drugs that may bind to conserved motifs in human homologs. The allosteric drugs bind to allosteric sites, induce c...
Developing allosteric drugs to treat pathogenic diseases can offer a promising alternative to orthosteric drugs that may bind to conserved motifs in human homologs. The allosteric drugs bind to allosteric sites, induce changes in the target protein's active site, and modulate its function with high selectivity, reduced adverse effects, and low toxicity. While identifying allosteric sites is costly and labor-intensive with experimental approaches, computational methods utilizing three-dimensional protein structures offer a cost-effective solution for discovering potential allosteric sites and predicting the effects of ligand binding. This study evaluates the effectiveness of two network models, the residue interaction network (RIN) model and the mixed coarse-grained anisotropic network model (mcgANM), in identifying putative allosteric regions, predicting the structural response of the protein to ligand binding, and elucidating allosteric mechanisms while maintaining computational efficiency. The SARS-CoV-2 main protease (M) is employed as an allosteric protein model due to a rich experimental and computational data available since the COVID-19 pandemic. The findings of the methods are assessed with statistical analysis, all-atom molecular dynamics simulations, and other elastic network models, namely Essential Site Scanning Analysis and Gaussian Network Model using a dataset of 15 ligand-bound and 4 ligand-free structures. RIN predicted the known drug binding sites of M with high statistics, up to 80.0% sensitivity, 89.7% specificity, 29.6% precision, and 89.2% accuracy. RIN suggested an allosteric mechanism of M that facilitates the allosteric communication of the allosteric and active sites through residue fluctuations. RIN was able to decompose the enzyme structure to dynamic domains, showing the organization of structural components to form a functional viral protease. mcgANM suggested the changes in residue fluctuations after ligand binding. The findings underscore the utility of the network models in advancing allosteric drug design.
Benchmarking Deep Learning for PROTAC Ternary Complex Prediction.
Chen H, Wei F, Li J … +8 more , Shi Z, Chen S, Fang Y, Li S, Gao X, Ju L, Fang S, Xu X
Proteins · 2026 Jul · PMID 41635150 · Publisher ↗
Proteolysis Targeting Chimeras (PROTACs) represent a transformative approach to drug development by leveraging the intracellular ubiquitin-proteasome system (UPS) for the selective degradation of target proteins. A PROTA...
Proteolysis Targeting Chimeras (PROTACs) represent a transformative approach to drug development by leveraging the intracellular ubiquitin-proteasome system (UPS) for the selective degradation of target proteins. A PROTAC molecule comprises three essential components: a ligand that binds to the E3 ubiquitin ligase, a ligand that targets the protein of interest, and a flexible linker that connects the two. This distinctive structure enables the PROTAC to simultaneously engage with both the target protein and the E3 ligase, facilitating their interaction. Such proximity initiates the ubiquitination of the target protein, marking it for recognition and subsequent degradation. In this study, we benchmark ternary complexes based on PROTACs using four recently employed predictive tools: Chai-1, AlphaFold2, AlphaFold3, and Protenix. Comparative analysis indicated that the ternary complexes predicted by the four prediction tools demonstrated satisfactory accuracy (Cα-RMSD < 10 Å). Among the evaluated tools, three-Chai-1, AlphaFold3, and Protenix-demonstrated superior performance in over half of the tests, while AlphaFold2 exhibited comparatively lower performance. However, significant challenges remained in accurately predicting the orientation of POI and the E3 ligase (Cα-RMSD < 10 Å when POI or E3 ligase were used as reference), as well as the position of the small molecule PROTAC (RMSD < 5 Å). By benchmarking these tools, we underscore recent advancements in protein structure prediction, enhance our understanding of the mechanisms underpinning PROTAC complexes, and provide a valuable reference for evaluating the binding conformations of other ternary complexes, as well as for the development of future predictive tools.
Identification and Characterization of Outer Membrane Proteins and Membrane Spanning Protein Complexes in Brucella melitensis.
Kapoor J, Panda A, Naqvi I … +3 more , Ganta S, Kumar S, Bandyopadhyay A
Proteins · 2026 Jul · PMID 41582728 · Publisher ↗
Brucellosis (Malta fever) is a zoonotic disease that affects both humans and animals, including cattle, sheep, and goats. Brucella melitensis is the most virulent and clinically significant species in humans. It is a gra...
Brucellosis (Malta fever) is a zoonotic disease that affects both humans and animals, including cattle, sheep, and goats. Brucella melitensis is the most virulent and clinically significant species in humans. It is a gram-negative bacterium with three groups of outer membrane proteins (OMPs): minor OMPs (Group 1) and major OMPs (Groups 2 and 3). OMPs with β-barrel architecture play important roles in nutrient transport, efflux, adhesion, and membrane biogenesis. Despite their importance, the structure, function, and interaction dynamics of several B. melitensis β-barrel OMPs and associated protein complexes remain mostly unexplored. In this study, we conducted a comprehensive in silico analysis to characterize known outer membrane β-barrel (OMBB) proteins and identify novel OMBBs in B. melitensis 16 M. Proteins were modeled using five computational tools: AlphaFold 3, ESMFold, SWISS-MODEL, RoseTTAFold, and TrRosetta. Outer-membrane insertion of 12 novel OMBBs was confirmed using PPM 3.0, Protein GRAVY, DREAMM, and MemProtMD_Insane. Putative functions were predicted using structure- and sequence-based annotations. Sequence variation across 46 B. melitensis strains was identified and mapped onto the structural models. OMBB-associated protein complexes-the RND (Resistance-Nodulation-Division) efflux pumps, the lipopolysaccharide transport (Lpt) complex, and the β-barrel assembly machinery (BAM) complex-were modeled, and protein-protein interactions (PPIs) were analyzed to confirm thermodynamically stable assemblies. This study presents a robust in silico strategy for exploring OMP architecture and provides valuable structural insights to support the development of diagnostics, targeted therapeutics, and vaccines against B. melitensis.
Improving Effector Protein Prediction in Phytoplasmas Through Structural Analysis of Signal Peptide Cleavage.
Derecik K, Tulum I
Proteins · 2026 Jul · PMID 41582677 · Publisher ↗
Phytoplasmas are highly destructive phloem-restricted pathogens, acting as obligate plant parasites transmitted by sap-feeding insect vectors. They infect over 1000 plant species, including critical crops, leading to sev...
Phytoplasmas are highly destructive phloem-restricted pathogens, acting as obligate plant parasites transmitted by sap-feeding insect vectors. They infect over 1000 plant species, including critical crops, leading to severe agricultural losses globally. Evolving from Gram-positive bacteria, phytoplasmas underwent extreme genome reduction, resulting in some of the smallest known bacterial genomes. Despite their minimal genetic content, they effectively manipulate host and vector cellular processes through effector proteins. These virulence factors are thought to be secreted via signal peptide (SP)-dependent cleavage by signal peptidase I (SPase I). Since phytoplasmas remain unculturable in vitro, identification of these effectors relies heavily on in silico SP and cleavage site (CS) prediction methods, which often produce unreliable results, leading to misidentified effector candidates. In this study, to improve prediction accuracy, we applied a structural modeling approach that complements sequence-based methods by assessing SPs through 3D modeling of SP-SPase I hetero-oligomer complexes. We analyzed reference virulence proteins (RVPs) with experimentally validated SPs, identifying potential errors in their annotated CSs. Through structural characterization, we classified phytoplasma SPase Is as eukaryotic ER-type-a rare trait in bacteria-and modeled SP-SPase I hetero-oligomers using ColabFold. Our findings reveal structural determinants governing cleavable SP binding to SPase I, enabling more accurate SP/CS predictions. This work underscores the unique molecular adaptations of phytoplasmas and provides insights for targeting their effector secretion mechanisms in disease control.
In Silico Functional and Structural Characterization of Streptococcus pneumoniae Atypical Rib Domain-Containing Hypothetical Protein Unravels Conserved Immunogenic Epitopes.
Arcan SKC, Gan GMD, Almazan DJN … +3 more , Duquilla ZN, Barbosa AVD, Velasco RUD
Proteins · 2026 Jun · PMID 41567112 · Publisher ↗
Streptococcus pneumoniae is a high-mortality pathogen exhibiting broad-spectrum antibiotic resistance, necessitating the development of alternative therapies, such as antigenic protein-based vaccines, which have recently...
Streptococcus pneumoniae is a high-mortality pathogen exhibiting broad-spectrum antibiotic resistance, necessitating the development of alternative therapies, such as antigenic protein-based vaccines, which have recently gained interest due to their novelty. Here, we characterized antigenic hypothetical proteins (HPs) of S. pneumoniae and determined their potential as vaccine construct targets. Subcellular localization reported 10 extracellular proteins, six of which were antigenic and nonallergenic, thus making them ideal vaccine construct targets. Functional annotation through conserved protein domain and motif prediction identified a unique, atypical Rib (aRib) domain from WP_001166178.1, widely distributed on bacterial cell surface proteins. A comparison with a canonical Rib domain showed domain atrophy, highlighting the lack of structural core elements. Further analysis revealed non-covalent interactions of Thr47, Ala48, Val41, and Phe38 interacting with an alpha-d-mannopyranose ligand, triggering S. pneumoniae colonization and capsule synthesis mechanism, with highly dynamic and flexible residues present on the ligand binding site. A strong immune response was observed from a computational immune response simulation, likely attributed to the presence of predicted 4 cytotoxic T lymphocyte (CTL), 10 helper T lymphocyte (HTL), and 5 B-cell lymphocyte (BCL) epitopes. Therefore, the study presents a novel protein for designing a vaccine construct against S. pneumoniae , thus offering a new target for future vaccinology studies. Future studies should confirm protective efficacy of this candidate in vitro and in vivo through immunological assays.
Integration of Evolutionary Analysis With RFdiffusion for De Novo Design of Aggregation-Resistant Frataxin.
Kırboğa KK, Küçüksille EU
Proteins · 2026 Jun · PMID 41563298 · Publisher ↗
Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by frataxin (FXN) deficiency, where protein replacement therapy is hampered by the inherent instability and aggregation propensity of wild-type (WT) FXN....
Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by frataxin (FXN) deficiency, where protein replacement therapy is hampered by the inherent instability and aggregation propensity of wild-type (WT) FXN. The structural flexibility of Loop-1 (residues 115-123), a critical region within the acidic ridge, represents a key determinant of protein stability. This study introduces a computational pipeline integrating evolutionary conservation analysis (ConSurf) with diffusion-based de novo design (RFdiffusion) to redesign both the backbone and sequence of Loop-1. Through systematic filtration of 1000 ProteinMPNN-generated variants using aggregation propensity screening (AGGRESCAN) and 450 ns of molecular dynamics (MD) simulations, four lead candidates were identified. Design_188 (EERVGGREI) demonstrated optimal performance with 2.3-fold improvement in aggregation resistance (Na4vSS: -53.8 vs. -23.5 for WT), superior structural stability (RMSD: 0.486 nm), reduced conformational diversity (62.3% dominant cluster occupancy), and 93% retention of ISCU binding capacity (ΔΔG: +6.4 kcal/mol). Experimental validation through N NMR relaxation analysis confirmed computational predictions, with Design_188 exhibiting uniform backbone rigidification (S = 0.81-0.95) and strong MD-NMR correlation (Pearson r = 0.675, p = 0.003). SEC-MALS analysis demonstrated near-complete monomeric behavior (> 98% monomer content) compared to WT's heterogeneous oligomerization (68% monomer, 32% oligomers), directly confirming the predicted anti-aggregation properties. K-means clustering analysis revealed an inverse relationship between conformational heterogeneity and stability, while correlation analysis identified a fundamental trade-off between aggregation resistance and structural stability (r = -0.82, p < 0.01). This work establishes a generalizable framework for therapeutic protein engineering where backbone redesign enables conformational ensemble modulation beyond the limitations of sequence optimization alone.
Snapshots of Motion: A Novel Structural Intermediate Reveals Conserved Dynamics in Archaeal DNA Ligases.
Quintana-Armas AX, Flores-Hernández E, Cardona-Félix C … +1 more , Rudiño-Piñera E
Proteins · 2026 Jun · PMID 41560473 · Full text
We present the first x-ray crystallographic structural evidence of an archaeal DNA ligase showing the AMP covalent adduct together with further cofactor hydrolysis, capturing a transient intermediary in the first step of...
We present the first x-ray crystallographic structural evidence of an archaeal DNA ligase showing the AMP covalent adduct together with further cofactor hydrolysis, capturing a transient intermediary in the first step of the ligation reaction, triggered by the pyrophosphate hydrolysis. Our crystallographic models of Thermococcus gammatolerans DNA ligase (LigTgam), coupled with bioinformatic analysis of at least 28 crystallographic structures from ATP- and NAD-dependent DNA ligases, highlight the central role of domain mobility. Notably, elevated B-values are consistently observed in key catalytic and binding regions, suggesting a link between structural flexibility and enzymatic efficiency. Remarkably, this pattern of high B-values is conserved in replicative ligases, including bacterial Lig A, indicating a broader evolutionary relevance. These fluctuations emphasize the importance of conformational adaptability in accommodating substrate DNA and facilitating catalytic steps, including adenylation and phosphodiester bond formation. In this work, we delve deeper into this dynamic behavior, providing evidence of its critical role in ligase function.
Allosteric Regulation of RNA Affinity by Motif V-VI Coupling in West Nile Virus NS3 Helicase.
Roy P, McCullagh M
Proteins · 2026 Jun · PMID 41558996 · Full text
The rise of flaviviral diseases, including West Nile virus (WNV), presents a growing threat to global public health and underscores the urgent need for new therapeutic strategies. The non-structural protein 3 helicase (N...
The rise of flaviviral diseases, including West Nile virus (WNV), presents a growing threat to global public health and underscores the urgent need for new therapeutic strategies. The non-structural protein 3 helicase (NS3h) of the Orthoflavivirus genus, including WNV, is essential for viral replication and a promising antiviral target. Previously [Roy et al., Nucleic Acids Research, 52 (13), 2024, 7447-7464], we showed that the motif VI loop (VIL) in WNV NS3h functions as a nucleotide valve, regulating ADP affinity during hydrolysis. In this study, we uncover an ATP-dependent coupling between nucleotide affinity at motif VIL and RNA affinity at motifs IVa and V, suggesting a coordinated mechanism of ssRNA translocation. Using microsecond-scale all-atom molecular dynamics simulations of hydrolysis-cycle intermediates, we find that key VIL residues (R461, R464) correlate strongly with RNA phosphate affinity of motif V. Structural analyses reveal an ATP-sensitive interaction between E413 (motif V) and R461 (motif VIL) that modulates the conformation of the motif V 3-helix, thereby influencing RNA binding. This dynamic interaction is lost in catalytically deficient VIL mutants, which have been experimentally shown to impair hydrolysis and attenuate viral replication. These findings provide mechanistic insights into NS3h function and identify new opportunities for structure-based antiviral design.
Structural and Biophysical Characterization of the Yersinia Type Three Secretion System ATPase YscN.
Barker SA, Ellis PK, Hammer A … +2 more , Johnson SJ, Dickenson NE
Proteins · 2026 Jun · PMID 41556233 · Full text
Yersinia pestis was responsible for the Black Plague, one of the worst epidemiological disasters in recorded history. Today, Y. pestis , Y. enterocolitica , and Y. pseudotuberculosis remain clinically relevant human path...
Yersinia pestis was responsible for the Black Plague, one of the worst epidemiological disasters in recorded history. Today, Y. pestis , Y. enterocolitica , and Y. pseudotuberculosis remain clinically relevant human pathogens. Each of these pathogenic Yersinia species relies on a Type Three Secretion System (T3SS) for virulence, with the ATPase YscN playing a critical role in T3SS function. T3SS ATPases are responsible for powering apparatus formation and effector protein secretion through ATP hydrolysis. This study provides an extensive enzymatic characterization of recombinant YscN under several conditions, including variable pH and temperature, substrate and protein concentrations, and in the presence of putative inhibitors. Thermal stability data, assessed by circular dichroism, demonstrate that YscN exhibits increased stability in alkaline conditions, coinciding with greatest ATPase activity. Further, we report the first high-resolution crystal structure of YscN and leverage homology data to model an oligomeric active site. Mutational analysis of a predicted active site residue confirms oligomerization as necessary for YscN ATPase activity and corroborates our oligomeric model and enzyme concentration-dependent specific activity. Interestingly, however, AUC analysis reveals that the purified YscN predominantly exists as a monomer, despite oligomerization-dependent active site formation. Thus, we propose that transient oligomeric interactions support the observed ATP hydrolysis. Together, these data uncover structural and environmental impacts on YscN activity that may support the highly specialized Yersinia pathogenic lifecycle and leverage its role in virulence in search of pan-effective small molecule T3SS ATPase inhibitors.
Identifying Bioactive Conformation of GnRH1 Based on Molecular Docking of NMR Conformers to GnRH1R and mAbs.
Bangaru MLY, Karande AA, Raghavendra NK
Proteins · 2026 Jun · PMID 41492967 · Publisher ↗
GnRH1 binds to its receptor GnRH1R to stimulate release of FSH and LH. Earlier NMR analysis had reported several possible conformers of GnRH1; however, the biologically active conformation of GnRH1 is not identified so f...
GnRH1 binds to its receptor GnRH1R to stimulate release of FSH and LH. Earlier NMR analysis had reported several possible conformers of GnRH1; however, the biologically active conformation of GnRH1 is not identified so far. Here, molecular docking of different NMR conformers of GnRH1 to GnRH1R is performed. Based on: (a) residues of GnRH1R interacting with antagonist elagolix (as ligand-binding pocket), (b) intermolecular hydrogen bonds (for specificity of interaction), and (c) total intermolecular non-covalent interactions (for stability of interaction), one NMR conformation, having an asymmetric U-turn reverse coil structure with a beta strand comprised of residues Gly6 and Leu7, is identified as the bioactive conformation of GnRH1. Further, the identified bioactive NMR conformation of GnRH1 is used to explain in vivo GnRH1-neutralizing ability of monoclonal antibody (mAb) F1D3C5 and lack of neutralization by another mAb E2D2H12. In mice, F1D3C5 completely blocks estrus cycle, while E2D2H12, despite having a relatively higher affinity for GnRH1 in ELISA, does not alter the estrus cycle. Molecular docking of the identified bioactive NMR conformation of GnRH1 to homology models of scFv attributes in vivo neutralizing ability of F1D3C5 to structure-specific recognition of GnRH1. The bioactive conformation of GnRH1 identified here could guide co-crystallization studies, design of analogs and GnRH1 vaccination efforts.
Structural Analysis and Inhibitor Modeling of Bacterioferritin From Brucella abortus.
Liu L, Harmon EK, Craig JK … +7 more , Yao H, Battaile KP, Johnson DK, Subramanian S, Van Voorhis WC, Rivera M, Lovell S
Proteins · 2026 Jun · PMID 41482512 · Full text
Iron homeostasis in various pathogenic bacteria is regulated by bacterioferritins (Bfr) which function to store Fe and release Fe as needed for metabolic processes. The Bfr structure consists of 18 kDa subunits in which...
Iron homeostasis in various pathogenic bacteria is regulated by bacterioferritins (Bfr) which function to store Fe and release Fe as needed for metabolic processes. The Bfr structure consists of 18 kDa subunits in which dimer pairs bind a heme molecule and are assembled into a highly symmetrical 24-meric spherical structure with an internal core diameter of approximately 80 Å. Release of iron is facilitated by the binding of a 7 kDa [2Fe-2S] ferredoxin (Bfd) to specific sites on the surface of Bfr which transfers electrons to the core thereby reducing the stored Fe to Fe for mobilization. The crystal structures of Bfr from Brucella abortus (Ba) in the apo and iron bound forms are presented and compared with those from Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa). Additionally, models of the Bfr:Bfd complexes for Ba and Ab are provided and compared with the Pa complex. Finally, compounds known to target the Bfr:Bfd interaction in Pa were docked to the Ba and Ab structures which provided insight regarding the potential binding mode and inhibitory mechanism.
Multi-Epitope Vaccine Design Against Leishmania donovani: An Immunoinformatic Based In Silico Approach.
Kaushik A, Pritam M, Govil S … +1 more , Kaushal RS
Proteins · 2026 Jun · PMID 41467395 · Publisher ↗
Leishmaniasis, caused by Leishmania donovani, remains a major neglected tropical disease (NTD) with limited therapeutic options and the absence of a universally effective vaccine. Multi-epitope vaccines offer a promising...
Leishmaniasis, caused by Leishmania donovani, remains a major neglected tropical disease (NTD) with limited therapeutic options and the absence of a universally effective vaccine. Multi-epitope vaccines offer a promising strategy for combating this intracellular parasite by stimulating a robust and specific immune response. In this study, an immunoinformatics-driven, in silico reverse vaccinology approach was utilized to design a multi-epitope vaccine targeting key surface-exposed proteins of L. donovani , namely C-type lectin, Proteophosphoglycan (PPG4), Hydrophilic Acylated Surface Protein (HASP), Legume-like Lectin (LLL), and Kinetoplastid Membrane Protein (KMP-11). These proteins are implicated in essential processes such as parasite survival, immune modulation, and host-pathogen interactions, making them prime candidates for vaccine development. A comprehensive analysis was conducted to identify and screen B-cell and T-cell (MHC-I and MHC-II) epitopes for immunogenicity, antigenicity, and population coverage. Multi-epitope vaccines, incorporating individual proteins or chimeric constructs, were developed with IFN-gamma as an adjuvant. The vaccine constructs were prioritized based on factors such as IC values and immunogenic potential. Subsequently, the selected epitopes were analyzed for physicochemical properties, and secondary and tertiary structural predictions were made and validated. Molecular docking simulations were employed to examine the interaction of the vaccine constructs with immune receptors, ensuring optimal immune system activation. Based on the molecular docking score, the vaccine candidates were screened and top four constructs (vaccines based on C-type lectin, LLL, PPG and chimeric vaccine; -1048.9, -1025.8, -1291.8, and -852.1 Kcal/mol respectively) were processed through immunogenic simulation. This in silico analysis indicates that lectins are highly effective vaccine candidates. Further, top two constructs, based on the immunogenic simulations, underwent molecular dynamics simulations. In the end, the final constructs were computationally cloned in pET28a vector. This study underscores the potential of multi-epitope vaccines as a cost-effective and efficient strategy for addressing L. donovani infections, providing a foundation for subsequent experimental validation and clinical trial development.
Navigating the Pre- and Post-AlphaFold Divide: CAPRI 8th Evaluation Meeting, February 12-14, Grenoble, FR.
Bonvin AMJJ, Lensink MF
Proteins · 2025 Dec · PMID 41431140 · Publisher ↗
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Mechanistic Insights Into the Inhibition of Dengue Virus NS5 Methyltransferase by Herbacetin.
Bhutkar M, Verma S, Singh V … +2 more , Kumar P, Tomar S
Proteins · 2026 May · PMID 41431129 · Publisher ↗
Herbacetin (HC) is a naturally occurring flavonoid compound with a dual antiviral mechanism. It inhibits the polyamine biosynthetic pathway and targets the methyltransferase (MTase) enzyme of both the dengue virus (DENV)...
Herbacetin (HC) is a naturally occurring flavonoid compound with a dual antiviral mechanism. It inhibits the polyamine biosynthetic pathway and targets the methyltransferase (MTase) enzyme of both the dengue virus (DENV) and chikungunya virus (CHIKV). However, the detailed inhibition mechanism of DENV-3 non-structural protein (NS5) MTase by HC remains unclear. This study provides structural insights into the inhibition mechanism of HC by analyzing the crystal structure of DENV-3 NS5 MTase complexed with HC and S-adenosyl-L-homocysteine. Structural analysis revealed that HC binds to the Cap 0-RNA site near the GTP binding site in the DENV-3 NS5 MTase. Additionally, the fluorescence polarization assay demonstrated that HC inhibits GTP binding with an inhibition constant (K) value of ~0.43 μM. This is one of the first studies that identify an inhibitor that targets the conserved RNA-binding region of NS5 MTase, suggesting its potential as a highly effective scaffold for broad-spectrum antiviral agents against orthoflaviviruses.
Computational Characterization of the Role of LEM2/LaminA Interactions on the Stability of BAF-Dimer Using Molecular Simulations.
Muthachikavil AV, von Appen A, Kühne TD
Proteins · 2026 May · PMID 41414748 · Full text
The effect of the presence of the BAF-binding LEM-domain and LaminA Ig-fold on the stability of the BAF dimer was studied qualitatively using non-equilibrium pull simulations and quantitatively through the calculation of...
The effect of the presence of the BAF-binding LEM-domain and LaminA Ig-fold on the stability of the BAF dimer was studied qualitatively using non-equilibrium pull simulations and quantitatively through the calculation of the potential of mean force profile along BAF-BAF separation distance. We find that hydrophobicity plays a significant role in stabilizing the BAF dimer when LEM-domain and LaminA are bound. The role of LEM-domain and LaminA in stabilizing the BAF dimer is explored by quantifying the strength of interaction between them, which are critical components of the nuclear lamina.
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