Abstact

FRIL is a legume lectin from the hyacinth bean that has broad-spectrum antiviral activity. A distinctive trait of FRIL among similar mannose/glucose-specific legume lectins is that FRIL shows specificity for complex type N-glycans. We postulate that an extended binding site on FRIL facilitates this ligand selectivity. Here, we show legume lectin carbohydrate recognition domain (CRD) loop B is the main determinant of complex versus high-mannose N-glycan specificity in FRIL and Concanavalin A (ConA), respectively. First, we find that the inactive precursors of recombinant FRIL (rFRIL) and proConA (rproConA) can be activated via deglycosylation. Secondly, the cryo-EM structures of inactive apo rFRIL, active FRIL in complex with Galbeta1,4-(Fucalpha1,3-)GlcNAcbeta1,2-Man tetrasaccharide, and active rFRIL in complex with Mannose(9)GlcNAc(2) (Man9) N-glycan are determined, and residues H102 and Y101 on loop B are identified as crucial for complex glycan recognition. Finally, we swapped loop B residues 101 and 102 alongside loop C residue 145 on FRIL to their structural equivalent on ConA, resulting in a FRIL mutant that binds exclusively to high mannose N-glycans. Taken together, we have established a process of activating recombinant FRIL and related lectins through deglycosylation, and demonstrated the crucial role that loop B residues play in establishing oligosaccharide specificity.