Lectins of the R-type superfamily recognize specific glycan motifs through β-trefoil carbohydrate-binding domains and play central roles in cell communication, immunity, and host-pathogen interactions. Here, we investigated the incompletely characterized glycan-binding specificity of Pleurocybella porrigens lectin (PPL) through an integrated structural approach combining NMR spectroscopy and X-ray crystallography. Three structurally related ligands of increasing biological complexity were analyzed: N-acetylgalactosamine (GalNAc), galactose (Gal), and galacto-N-biose (Galβ1 → 3GalNAc), a minimal mucin-type O-glycan epitope associated with cancer-related glycosylation. STD NMR experiments defined the ligand-binding epitopes in solution and revealed different contributions of individual sugar protons to PPL recognition. GalNAc displayed an extensive interaction profile, with the N-acetyl substituent playing a key role in stabilizing binding; whereas, in the case of Gal, PPL showed an exquisite preference for the recognition of the β-anomer. X-ray crystal structures, obtained by soaking and co-crystallization experiments, provided atomic-resolution insight into ligand accommodation within the β-trefoil binding pocket, identifying hydrogen-bonding networks and apolar interactions responsible for glycan recognition. The combined NMR and crystallographic data establish consistent models for PPL binding to various ligands. Our findings clarify the structural basis of Gal/GalNAc selectivity in PPL and support the broader utility of ricin-B-like lectins as probes for biologically and clinically relevant O-glycan epitopes. More generally, this work highlights the complementarity of NMR and X-ray crystallography for the integrated characterization of lectin-glycan interactions.
Angela Marseglia, Gabriella Tito, G. Ferraro et al.· International Journal of Bio...· 0 citations
Paddlewheel diruthenium (Ru2) complexes are promising modulators of protein aggregation due to their tunable coordination chemistry and dual-action properties. Here, we investigate the interaction of five Ru2 complexes with hen egg white lysozyme (HEWL), an amyloid model, to elucidate their antiaggregation capabilities. High-resolution X-ray crystallography shows that all complexes preferentially bind to Asp119 and, in some cases, Asp101, through coordination to the Ru2 core while preserving the overall protein fold. Both covalent and noncovalent interactions are observed, depending on ligand environment and steric effects. Solution studies confirm the formation of HEWL–Ru2 adducts under both neutral and acidic conditions. Functional assays demonstrate that all Ru2 complexes effectively inhibit HEWL fibrillogenesis, as indicated by reduced ThT fluorescence and the absence of large aggregates in dynamic light scattering measurements. Disaggregation of preformed fibrils was more variable, with the complex bearing vacant axial sites showing the highest activity. Circular dichroism and scanning electron microscopy analyses reveal that these compounds redirect aggregation toward noncanonical morphologies rather than fully dissolving fibrils. Cytotoxicity assays confirm reduced HEWL-induced cellular toxicity. Overall, our findings establish a correlation between ligand composition, coordination behavior, protein binding, and antiamyloid activity, providing a framework for designing Ru2-based multifunctional modulators of protein aggregation.
S. La Manna, Daniele Florio, G. Ferraro et al.· Inorganic Chemistry· 0 citations
Thioredoxin reductase from Burkholderia cenocepacia (Bc‐TrxR) is a recognized intracellular target of antibacterial gold(I) compounds, yet the molecular basis of enzyme inhibition remains unclear. Here, we report an integrated structural, biophysical, and biochemical investigation of recombinant Bc‐TrxR and its interaction with three prototypical gold(I) agents: auranofin (AF) and two trimethylphosphine‐thiolate derivatives (Au1 and Au2), previously identified as potent enzyme inhibitors. The enzyme was expressed, purified, and its crystal structure solved at 2.52 Å resolution, revealing a homodimeric architecture closely resembling that of the Escherichia coli enzyme. Each subunit contains FAD‐ and NADPH‐binding domains and a catalytic Cys–Cys motif representing a plausible coordination site for gold fragments. High‐resolution ESI‐MS provided direct molecular evidence for the formation of defined gold‐protein adducts, with up to two Au(I) centres bound per subunit. The observed mass shifts are consistent with selective coordination of gold fragments to catalytically relevant cysteine residues, effectively blocking redox turnover. Together, crystallographic and mass spectrometric results disclose the molecular mechanism of Bc‐TrxR inhibition by phosphine‐thiolate gold(I) complexes and establish a structural framework for the rational design of next‐generation antibacterial metallodrugs.
Stefano Zineddu, José Aleixo de Azevedo-França, Martina Aguanno et al.· ChemBioChem· 0 citations