Identification and characterization of novel hepcidin mimetics that modulate ferroportin function

Insulin-like growth factor 2 (IGF2) engages multiple receptors of the insulin-hormone family, yet the structural determinants of its selectivity remain poorly defined. Vesiculin, a naturally occurring des[37-40]IGF2 isoform, represents a unique probe for dissecting these interactions. Here, we combine chemical synthesis, receptor-binding assays, phosphorylation studies, and molecular dynamics simulations to characterize vesiculin and compare it with wild-type IGF2. Vesiculin consistently displayed a reduced affinity for the majority of binding partners, with the most pronounced loss observed for IGF2R and its isolated domain 11 (D11). Computational analyses revealed that this reduction originates from the destabilization of the IGF2 C-domain, which in wild-type IGF2 forms cooperative contacts with IGF2R domains D6 and D8. Deletion of Arg37-Arg40 abolishes these interactions and enforces ineffective compensatory contacts. For the insulin receptor, the loss of these residues disrupts stabilizing electrostatic contacts with the α-CT and L2 domains. In contrast, IGF1R binding is only modestly affected, and vesiculin shows a markedly diminished affinity for IGFBP3, raising questions about its bioactivity. Together, these findings redefine the IGF2-IGF2R binding interface and highlight the IGF2 C-domain as a central determinant of receptor specificity across the insulin-IGF system, providing a framework for designing IGF2 analogs.

BVDV NS5A promotes mitochondria-associated endoplasmic reticulum membrane (MAM) formation to enhance viral replication.

Protein-carbohydrate interactions play a key role in numerous biological processes, including immune response, and glycan-based ligands that can target specific protein receptors on a cell surface represent promising candidates for therapeutics applications. For example, in retinoblastoma, the DC-SIGN mannose receptor is overexpressed on the surface of pathogenic cells and represents an interesting target for mannose-based ligands. At the same time, these ligands should not bind to the MRC1 receptor, which is expressed by adjacent, healthy, retinal pigment epithelial cells and presents a carbohydrate recognition domain (CRD) similar to the one of DC-SIGN. Therefore, the challenge remains to obtain a detailed picture of the recognition process between carbohydrates and proteins, in order to design effective and selective therapeutic compounds. In this work we used classical, all-atom molecular dynamics (MD) simulations to investigate the interaction between several mannose based ligands and the CRDs from DC-SIGN and MRC1. The analysis of the protein-carbohydrate contacts from the resulting trajectories highlights the variability of the mannose binding modes on both CRDs, and shows how the increased affinity of mannose for the MRC1 CRD can be related to a specific mannose binding state that is not accessible in the DC-SIGN CRD.

A series of 2-phenylindoles were synthesized and studied the radical quenching properties towards compounds with nitrogen radical. The present indoles exhibit radical quenching properties in a substituent dependent manner through proton and electron transfer mechanisms. ortho-Hydroxy-2-phenylindole and para-hydroxy-2-phenylindole show strong radical quenching properties towards DPPH radical as comparable to vitamin C, whereas meta-hydroxy-2-phenylindole and 2-phenylindole exhibit radical quenching properties with lower efficacy. All these indoles, however, exhibit efficient radical quenching properties towards ABTS radical as comparable to DPPH radical (IC50 towards DPPH/ABTS: 1: 335/22 µM, 2: 15/7 µM, 3: 102/8 µM, 4: 9/10 µM, vitamin C: 10/3 µM). These compounds also bind to lipoxygenase (LOX) and inhibit LOX activity with IC50 : 18-44 µM. It is shown that O-H bond dissociation energy and ionization energy play important role in deciding the radical quenching properties of present indole compound. The compound-LOX binding affinity and binding interface is computed through autodock vina. The indole-LOX binding interface comprises of hydrophobic amino acid residues. In the presence of. indole molecule, the radical quenching occurs through hydrogen atom and/or through electron and subsequently proton transfer mechanism.

Therapeutic antibodies face on-target/off-tumor toxicity, causing adverse effects such as cardiotoxicity, skin rashes, and organ inflammation. To mitigate these challenges, pH-dependent antibodies have been engineered to preferentially bind in the acidic tumor microenvironment while reducing interactions under physiological pH conditions. Building on experimental work that generated Ipilimumab variants (Ipi95, Ipi105, Ipi106) through charged amino acid substitutions in complementarity-determining regions, we employed molecular dynamics simulations to examine their interactions with CTLA-4 in physiological and acidic conditions. All variants exhibited enhanced binding affinity at acidic pH, with a reasonable agreement between computational and experimental binding free energies (R2 = 0.7736; Pearson's r = 0.8795, p = 0.0039; Spearman's ρ = 0.8333, p = 0.0102). Statistical analysis revealed notable differences across conditions, most notably for Ipi95, which demonstrated the highest degree of pH sensitivity. Although no major global structural changes were observed between conditions, our simulations revealed distinct local energetic rearrangements and residue-level interaction changes at the binding interface. Decomposition analysis on binding energy further indicated that the overall antigen-binding mode was maintained, whereas the introduced charged residues modulated local interaction strengths. These results provide mechanistic insights into how targeted mutations modulate pH-dependent recognition, offering a framework for the rational design of safer therapeutic antibodies.

Immobilized recombinant FcγRIIIa and FcRn receptors as a tool for characterization of human IgG antibodies.

Excessive sugar intake remains a major health challenge, motivating the development of safe and effective alternatives. Thaumatin, a natural high-intensity sweet protein, elicits sweetness through activation of the sweet taste receptor (T1R2/T1R3), yet its molecular recognition mechanism remains understudied. An integrated computational strategy combining comparative modeling, protein–protein docking, and 500 ns molecular dynamics simulations (triplicates) was employed to elucidate the thaumatin–receptor binding. Structural modeling identified the closed conformation of the Venus flytrap domain (VFT) as optimal for ligand engagement. Modeling revealed a stable binding interface characterized by electrostatic complementarity and van der Waals interactions, characterized by interfacial contacts of receptors and hydrogen bonding networks. Residue-level energy decomposition highlighted key residues (W418 and E422 of T1R2; S59 of T1R3) and thaumatin residues (K67, R82, and K137) that contribute substantially to complex stabilization, consistent with experimentally reported sweetness determinants. These findings provide molecular-level insight into sweet protein recognition and establish a structural framework for rational engineering of protein-based sweeteners with enhanced potency and selectivity.

Structure of the IgY-FcRY complex and its comparison with IgG-FcRn.

NMR characterization of the structure and interaction of an RNA aptamer targeting α-synuclein.

During mitosis, properly aligned chromosomes stabilise microtubule ends with the help of kinetochores to ensure timely segregation of chromosomes. Microtubule-binding components of the human outer kinetochore, such as Ndc80 and Ska complexes, are present in multiple copies and together bind several microtubule ends, creating a highly multivalent binding interface. Whereas Ndc80:Ndc80 and Ndc80:microtubule binding is crucial for interface stability, Ndc80 alone in absence of Ska is unable to support stable kinetochore-attachments. Using cryo-electron tomography, we demonstrate that oligomeric Ndc80:Ska assemblies stabilise microtubule ends against shortening by strengthening lateral contacts between tubulin protofilaments at microtubule plus-ends. We further identify a point mutation within the SKA1 microtubule-binding domain that does not affect microtubule-binding of individual Ska molecules, but does abolish Ska:Ska interactions. Finally, we report that oligomerisation of Ska, in a cooperative fashion together with the Ndc80, is necessary to maintain stable microtubule attachments both in vivo and in vitro.

Cofilin is a key regulator of actin dynamics that, along with a myriad of other actin-binding proteins, controls the balance of F- and G-actin in numerous cell types. While prior structural studies of the cofilin-actin binding interface have delineated many critical interactions between cofilin and actin, the roles of some residues within the cofilin-actin binding interface remain poorly defined. In this study, we investigate the role of cofilin S119 in the cofilin-actin interaction. Despite its unique position within the cofilin-actin interface and its putative role as a phosphorylation site, relatively little direct evidence exists to define whether it plays an important role in cofilin-actin dynamics. Using site-directed mutagenesis, we demonstrate that mutation of S119 to aromatic amino acids (W, F, Y) results in cofilins with strong actin bundling activity in living cells. This activity can be countered by the incorporation of mutants that disfavor actin rod forming activity (R21Q). Mutation of S119 to phospho-mimic (E) and non-phosphorylated (A) residues either strongly inhibits (E) or modestly increases (A) actin bundling activity. Expression of the S119W mutant in neurons reveals its impacts on spine length and size, while FRAP studies show that its mobile fraction is intermediate between that of LifeAct and WT cofilin. Finally, it is shown that the strong actin bundling phenotype associated with S119W inhibits the progression of optogenetically induced apoptosis.

Eukaryotic cap-dependent translation initiation is regulated by binding of the predominantly folded eukaryotic initiation factor 4E (eIF4E) to the intrinsically disordered eIF4E binding proteins (4E-BPs). Here, we report full-length atomistic conformational ensembles generated by IDPConformerGenerator and optimized by X-EISDv2 workflow for both apo 4E-BP2, the neuronal 4E-BP, and 4E-BP2 in complex with eIF4E, using data from single-molecule fluorescence and nuclear magnetic resonance (NMR), together with select coordinates from a 4E-BP1:eIF4E crystal structure. Structural sampling within dynamic complexes is often under-appreciated, with NMR and crystal structure data for 4E-BP:eIF4E suggesting different degrees of structural heterogeneity. Our ensemble models validated by solution spectroscopy data enable comparison of free 4E-BP2 and its complex with eIF4E. This shows a delocalization of contacts around canonical regions, which supports previous findings of unidirectional conditional occupancy of the binding sites. Two new contact regions emerged: one between the disordered N-termini of eIF4E and 4E-BP2, which may play an allosteric role in tuning the binding affinity, and the other between the C-terminus of 4E-BP2 and an extended region of eIF4E, which is consistent with the extended, dynamic binding interface that we reported previously. These results support a model of translation regulation in which the dynamic 4E-BP2:eIF4E complex facilitates accessibility of regulatory sites of 4E-BP2 when bound.

Long-range communication in proteins is key to biological regulation and the efficacy of many drugs. However, the extent to which indirect energetic couplings between distant sites are conserved in homologous proteins during evolution is largely unknown. Here we directly address this question by constructing seven comprehensive maps of how mutations throughout five homologous human PDZ domains directly and indirectly alter the energy of binding to peptide ligands. The combined dataset quantifies 21,802 free energy changes - 9064 changes in fold stability (∆∆Gf) for five PDZ domains and 12,738 changes in binding energy (∆∆Gb) across seven interactions. The maps allow a comparison of the energetic landscapes of binding interfaces and the conservation of hotspot residues. They also allow comprehensive identification and comparison of allosteric mutations, those outside of the binding interface that alter the binding energy. The proteins have a conserved distant-dependent decay in the energetic effects of mutations away from the binding interfaces. However each protein also has protein-specific allosteric mutations, including in both structurally-aligned sites and protein-specific domain extensions. The divergence in the location of allosteric mutations in each protein suggests that each protein in a family might have distinct sites to target with allosteric drugs.

Inhibition of vascularendothelial growth factor 165 (VEGF165)-mediated angiogenesisis a promising anticancer strategy.While gold nanoparticles (AuNPs) are effective inhibitors of VEGF165, the precise molecular interface of their interactionswith VEGF165 is unclear, hindering rational therapeuticdesign. This study combined molecular dynamics (MD) simulations andexperimental techniques to map the binding interface between AuNPsand VEGF165. We discovered that AuNPs preferentially bindto the HQGQH motif on VEGF165. Functional assays showedthat this binding inhibited VEGF165-induced endothelialcell migration and angiogenesis. The necessity of the HQGQH motifwas unequivocally demonstrated by mutagenesis. A 5A-substituted VEGF165 mutant exhibited markedly reduced AuNP binding affinityacross binding free energy calculations, ELISA, and ζ potentialmeasurements, and its angiogenic activity became resistant to AuNPinhibition to some extent. These findings reveal the molecular basisof AuNP-mediated VEGF165 blockade, opening avenues fordesigning targeted antiangiogenic nanomedicines.

Allosteric effects are critical for protein function. The mechanisms by which allosteric effects propagate in cellular environments remain intriguing yet unresolved questions. In this study, we investigated the allosteric effects involved in a protein-protein binding process within Escherichia coli cells. Mutations far away from the binding interface were introduced, which alter the affinity through allosteric effects. The results indicate that entropy contributes favorably to the allosteric effect in cellular contexts. This entropy-driven allosteric effect is also corroborated by molecular dynamics (MD) simulations, which underscore the significance of the protein solvation water. The crowded macromolecules help to restrain solvation water, and the release of these water molecules creates a positive entropic gain, which in turn generates an allosteric effect in cells.

The chromatin reader ZMYND8 recruits the NuRD component GATAD2A through its MYND domain to regulate MAPT213 long noncoding RNA transcription.

Galectin-3 (Gal3) is one of the most pro-inflammatory proteins and a biomarker of inflammatory diseases and cancer. Previous studies showed that Gal3 binds to αv and β1 integrins, but it is unclear how Gal3 binds to integrins. Here, we show that Gal3 bound to soluble αvβ3 and αIIbβ3 integrins in 1 mM Mn2+ in cell-free conditions in a glycan-independent manner. Docking simulation predicts that Gal3 binds to the classical RGD-binding site (site 1) of αvβ3, but the predicted Gal3-binding site does not include galactose-binding site. RGDfV or eptifibatide inhibited Gal3 binding to αvβ3 and αIIbβ3, respectively, but lactose, a pan-galectin inhibitor, did not inhibit Gal3 binding to integrins. Point mutations of the predicted site 1 binding interface of Gal3 effectively inhibited Gal3 binding to site 1. Site 2 is involved in pro-inflammatory signaling (e.g., TNF and IL-6 secretion), and we previously showed that pro-inflammatory cytokines (e.g., CCL5 and TNF) bind to site 2 and allosteric integrin activation. Docking simulation predicted that Gal3 binds to site 2 of αvβ3 and α5β1. We found that Gal3 induced allosteric activation of soluble integrins αvβ3, αIIbβ3, and α5β1 in 1 mM Ca2+ in cell-free conditions. Point mutations in the predicted site 2 binding interface inhibited Gal3-induced integrin activation, suggesting that Gal3 binding to site 2 is required for Gal3-induced integrin activation. Known anti-inflammatory agents, Ivermectin, NRG1, and FGF1, inhibited integrin activation induced by Gal3 in αvβ3 and αIIbβ3. These findings suggest that Gal3 binding to site 2 may be a potential mechanism of pro-inflammatory and pro-thrombotic action of Gal3.

Non-vesicular lipid transport contributes to the regulation of membrane composition and organelle function at membrane contact sites. OSBP-related proteins (ORPs) are central to this process, yet their interaction networks remain incompletely defined. Here, we systematically screened potential interactions between ORPs and phosphoinositide 3-, 4-, and 5-phosphatases using AlphaPulldown2, AlphaFold2-Multimer, and AlphaFold3. We established a protocol for model generation by combining AlphaFold2-Multimer predictions (including five-replicates) with an AlphaPulldown2 interaction screen across around 200 protein pairs, and with AlphaFold3 predictions including lipid-bound and multimeric assemblies. Interface confidence was assessed for consistency using the weighted ipTM + pTM metric, actifpTM, new generation ipSAE scoring, and FoldSeek-Multimer clustering. We further evaluated the protein pairs' biological plausibility based on subcellular localization data, in silico membrane insertion, evolutionary conservation via ConSurf, and protein binding interface analysis using the deep learning tool PeSTo. This integrative protocol uncovered functionally conserved binding modes in the SAC1 lipid phosphatase with the ORP family, particularly with ORP11, and predicted functionally relevant protein-lipid interfaces.

Rademikibart (CBP-201) is a human monoclonal antibody with higher binding affinity to IL-4Rα compared to dupilumab. Dupilumab is a first-generation interleukin-4 receptor alpha (IL-4Rα) inhibitor for treating IL-4Rα-dependent inflammatory disorders, including several dermatologic and respiratory conditions. Rademikibart, however, demonstrated better inhibition of STAT6 intracellular signaling in vitro and similar potency in inhibiting both IL-4 induced TARC release and IL-4 induced B cell activation. To further characterize the molecular function of rademikibart and its differentiation from dupilumab, we determined the crystal structure of the rademikibart fragment antigen binding (Fab) bound to IL-4Rα at 2.71 Å resolution and compared this to the 2.82 Å resolution structure of dupilumab Fab bound to IL-4Rα. The rotation angle between dupilumab and rademikibart bound to IL-4Rα is 54.88°. This rotation enables the binding epitopes of rademikibart, but not dupilumab, on IL-4Rα to overlap more closely with the conserved binding interface naturally utilized by IL-4 and IL-13 cytokines. Molecular dynamics (MD) studies on rademikibart and dupilumab bound to IL-4Rα examined the stability of the complexes and effects of amino acid mutations on receptor complex formation. MD simulations demonstrated that the third interface loop (residues 145 to 153 in domain 2) of IL-4Rα interacts directly with rademikibart, which is absent in the dupilumab/IL-4Rα complex. This finding is confirmed by increased hydrogen bond interactions at the interface between rademikibart and IL-4Rα, demonstrating superior binding energy for rademikibart. Through analysis of the x-ray crystallography structures, MD-equilibrated structures, and computational point-mutation analysis of rademikibart, we identified residue Y50 and R55 of the light chain and R97, R99, and Y101 of the heavy chain of rademikibart as key residues interacting with IL-4Rα's third interface loop. Our data provides a molecular and structural rationale for the enhanced IL-4Rα inhibition by rademikibart over dupilumab, confirming rademikibart as an optimized second-generation IL-4Rα inhibitor.