Ezrin, a key member of the ERM (Ezrin–Radixin–Moesin) family, links the plasma membrane to the actin cytoskeleton and is regulated by a conformational switch between closed and open states. Although phosphatidylinositol 4,5-bisphosphate binding and phosphorylation at Thr567 are known to promote Ezrin activation, the associated conformational mechanism remains incompletely understood. Here, we combined Martini coarse-grained molecular dynamics simulations with enhanced sampling, all-atom molecular dynamics simulations, and MM/PBSA calculations to compare wild-type Ezrin with the phosphomimetic T567D mutant. The simulations indicate that T567 phosphorylation destabilizes the FERM/C-ERMAD interface, promotes domain separation, and facilitates exposure of membrane- and actin-binding sites. Computational analysis of binding free energies further demonstrates that the interaction between FERM and C-ERMAD is attenuated due to the phosphorylation of T567. Finally, a stepwise dissociation mechanism for the C-ERMAD α-helices is proposed. These results support a model in which T567 phosphorylation primarily promotes the initial transition toward an open conformation, whereas complete unfolding and full activation likely require additional events, including PIP2 binding, recruitment of partner proteins, and interaction with actin filaments.
Gaucher disease (GD) is a lysosomal storage disorder caused by deficient acid β-glucosidase (GCase) activity and consequent accumulation of glucosylceramide. Current enzyme replacement and substrate reduction therapies improve several systemic manifestations but have limited impact on neuronopathic disease, motivating evaluation of alternative molecular strategies. This study computationally assessed the interaction of EPA-phosphatidylcholine (EPA-PC), a marine-derived omega-3 phospholipid, with human GCase (PDB ID: 2NSX). The protein structure was examined by Ramachandran analysis and hydrophobicity profiling, followed by AutoDock Vina engine docking at the proposed hydrophobic i-face. EPA-PC showed a docking score of -5.5 kcal/mol under the applied protocol. Independent PLIP profiling of the docked complex identified predominantly hydrophobic contacts, one hydrogen bond involving Phe347, and Arg395-mediated salt-bridge interactions. A single 200 ns molecular dynamics trajectory of the EPA-PC-GCase complex showed an apparent backbone RMSD plateau of approximately 0.35-0.40 nm and a radius of gyration near 2.4 nm without a sustained increase in global compactness or solvent exposure. These observations support persistence of the modelled complex during the reported trajectory but do not establish ligand-induced GCase stabilisation or pharmacological-chaperone activity. In silico ADMET profiling also indicated substantial liabilities, including very high plasma protein binding and predicted hERG-related risk. EPA-PC should therefore be regarded as a preliminary lipid-protein interaction model requiring comparative simulations, independent replicates, and experimental validation.
Human epidermal growth factor receptor 2 (HER2) is an oncogenic receptor tyrosine kinase in breast cancer and other malignancies. A subset of HER2-positive tumours expresses 611-CTF-p95HER2, a tumour-specific, hyperactive truncated isoform associated with metastasis and treatment resistance that lacks most of the extracellular domain targeted by conventional HER2-directed antibodies. We previously developed NAZ-mAb (formerly known as Oslo-2), a monoclonal antibody against 611-CTF-p95HER2. Here, we describe a computational antibody-engineering workflow for designing variants of NAZ-mAb. Starting from the sequence alone, we modelled the NAZ-mAb–611-CTF-p95HER2 complex, generated a combinatorial mutational landscape using FoldX 5.0, and prioritized candidate variants using predicted interaction energy and developability criteria. Two variants representing distinct design strategies were selected for validation: an aromatic double mutant, NAZ-mAb v1 (L:S31W/L:H107W), and a conservative single mutant, NAZ-mAb v2 (L:S31M). Both variants were successfully expressed as recombinant IgGs; NAZ-mAb v2 achieved a five-fold higher recombinant expression yield than parental NAZ-mAb, while both variants retained antigen binding with a higher apparent signal than the parental antibody in indirect ELISA. However, Biacore two-state kinetic analysis revealed weaker affinities than the parental antibody (KD NAZ-mAb v1: 326.1 nM, NAZ-mAb v2: 94.5 nM vs parental NAZ-mAb: 53.3 nM). These findings show that the computational workflow can generate antigen-engaging NAZ-mAb variants, while also highlighting the limitations of fixed-backbone interaction-energy ranking as a predictor of binding affinity and yield. This study provides a practical framework and a cautionary benchmark for computationally driven, developability-aware antibody engineering in the absence of experimental structural data.
WNTs are a family of signaling proteins involved in numerous biological processes, including morphogenesis, oncogenesis, cell migration and proliferation, cellular specialization, and tissue regeneration. WNT proteins are characterized by a distinctive structure comprising two domains resembling a "thumb" and an "index finger," which enables their interaction with cellular receptors. Despite the identification of 19 WNT proteins in humans, three-dimensional structural data are available for only a subset, highlighting the importance of studying WNT proteins using in silico approaches. This study presents the in silico structural analysis of three human WNT family members - WNT1, WNT3A, and WNT5A - across multiple levels of protein organization. The primary structure of these proteins was analyzed for amino acid composition, and secondary structure predictions for α-helices, β-strands, and loops were correlated with domain structures characteristic of WNT proteins. Tertiary structures were modeled using homology modeling and deep-learning algorithms, and structural properties were investigated through molecular dynamics simulations. The ability of WNT proteins to adopt several closed and open conformational states was demonstrated, as well as the role of the flexibility of the β-hairpin forming the "index finger" in mediating conformational transitions. Thus, by applying a comprehensive bioinformatic approach to WNT proteins, the study highlights the advanced capabilities of in silico methods for analyzing protein structures at multiple levels of organization.
The enzymes Cannabichromenic Acid Synthase (CBCAS), Cannabidiolic Acid Synthase (CBDAS) and Tetrahydrocannabinolic Acid Synthase (THCAS) are together the major cannabinoid synthase enzymes responsible for the biosynthesis of their respective metabolites from a common precursor Cannabigerolic Acid (CBGA). As the catalysts responsible for generating biological molecules of significant pharmaceutical value, there has been considerable interest in the enzymes with respect to heterologous production, mechanism, and incorporation into synthetic biology pathways for the facile industrial production of these molecules. The enzymes share high degrees of homology, and therefore their distinct specificities are governed by very subtle differences in sequence and therefore structure, although, until now, only a structure for THCAS has been reported. In this report, we present structures of CBCAS, CBDAS and a structure of THCAS at a higher resolution than the known structure, each in complex with their flavin coenzyme FAD. The structures reveal active site differences that may be responsible for the complementary activities observed, in terms of both first-shell amino acid substitutions, but also in more remote residues that influence active site topology through referred effects, or that have effects on substrate access. The structures provide a useful and informative platform for the rational engineering of improved or altered chemoselectivity in these enzymes.
