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
The global rise of multidrug-resistant bacteria is a major health threat, with Klebsiella pneumoniae identified by the WHO as a critical priority pathogen. Phage-derived depolymerases have emerged as promising countermeasures because they can degrade the capsular polysaccharide (CPS) that shields bacteria from the immune system, thereby increasing their susceptibility to antibiotics and host defences. However, the limited understanding of how they recognise specific CPS structures remains a major obstacle to developing effective depolymerase-based therapeutics. In this study, we provide a comprehensive NMR and mutational analysis to characterise the mechanism of action of a miniaturised depolymerase, here mKP34gp57, targeting the clinically relevant K63 CPS. We show by NMR that mKP34gp57 hydrolyses CPS with high efficiency through an endoglycosidase-retaining mechanism. During substrate recognition, our data demonstrate that the enzyme interacts predominantly with the galactose and fucose moieties, which serve as the critical recognition features and thus the principal determinants of CPS specificity. Computational studies provide structural clues for the roles of the catalytic residues E266/E300 and D151. Finally, we prove that the hexasaccharide produced upon CPS hydrolysis stimulates dendritic cell maturation and T-helper-driven lymphocyte proliferation. Identifying the binding determinants that govern CPS recognition by mKP34gp57, and using this information to generate immunogenic fragments, deepens our understanding of how minidepolymerases can be rationally engineered to achieve tailored serotype specificity, and improved therapeutic and diagnostic potential.
Ferran Nieto-Fabregat, Mario Privitera, Angela Marseglia et al.· International Journal of Bio...· 0 citations
