Unveiling the molecular basis of Pleurocybella porrigens lectin (PPL)-glycan recognition by NMR and X-ray crystallography.

Abstract

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.