Structural Insights into Recognition of Triple-helical β-Glucans by an Insect Fungal Receptor

Abstract

The innate ability to detect pathogens is achieved by pattern recognition receptors, which recognize non-self-components such as ␤1,3-glucan. ␤1,3-Glucans form a triple-helical structure stabilized by interchain hydrogen bonds. ␤1,3-Glucan recognition protein (␤GRP)/Gram-negative bacteria-binding protein 3 (GNBP3), one of the pattern recognition receptors, binds to long, structured ␤1,3-glucan to initiate innate immune response. However, binding details and how specificity is achieved in such receptors remain important unresolved issues. We solved the crystal structures of the N-terminal ␤1,3-glucan recognition domain of ␤GRP/GNBP3 (␤GRP-N) in complex with the ␤1,3-linked glucose hexamer, laminarihexaose. In the crystals, three structured laminarihexaoses simultaneously interact through six glucose residues (two from each chain) with one ␤GRP-N. The spatial arrangement of the laminarihexaoses bound to ␤GRP-N is almost identical to that of a ␤1,3-glucan triple-helical structure. Therefore, our crystallographic structures together with site-directed mutagenesis data provide a structural basis for the unique recognition by such receptors of the triple-helical structure of ␤1,3-glucan.Carbohydrate polymers form a wonderful variety of shapes and architecture with their different glycosidic linkages and many hydrogen bonding possibilities, as exemplified by the glucose polymers, cellulose, amylose, and ␤-glucan (1). ␤-Glucan, mostly found in fungi and plant cell walls, is a polymer consisting of a backbone of ␤1,3-linked glucose units and side chains of ␤1,6-linked units of varying lengths. ␤1,3-Glucan assumes a triple-helical structure according to x-ray fiber diffraction (2), solid state 13 C nuclear magnetic resonance spectroscopy (3), multiangle laser light scattering (4), fluorescence resonance energy transfer spectroscopy (5), and molecular dynamics simulation (6). In a widely accepted hydrogen bonding model, one ␤1,3-glucan chain forms intramolecular hydrogen bonds with the other two strands, perpendicular to the axis of the triple helix (7).The ␤-glucan of pathogens is recognized as non-self by pattern recognition receptors in the host defense system (8 -11). Accumulating evidence suggests that the long triple-helical conformation of ␤-glucan is important for binding to certain ␤-glucan-binding receptors (7, 12). ␤1,3-Glucan recognition protein/Gram-negative bacteria-binding protein 3 (␤GRP/ GNBP3) 3 (13-15), a pattern recognition receptor of the innate immune system, binds triple-helical ␤-glucan strongly but has little affinity for denatured ␤-glucan or shorter ␤1,3-linked glucose oligomers (16,17). Furthermore, injection into the fly, Drosophila, of the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which includes long ␤1,3-glucans, strongly activates the antifungal Toll pathway (14), whereas short laminarioligosaccharides do not (16). To date, three-dimensional structures of the ligand-free ␤GRP/GNBP3 N-terminal domain were reported revealed by x-ray crystallography (16) and solution NMR...

Self-association of an Insect β-1,3-Glucan Recognition Protein Upon Binding Laminarin Stimulates Prophenoloxidase Activation as an Innate Immune Response

“…In contrast to insoluble linear polysaccharides such as curdlan and cellulose, laminarin is soluble in water, because its ␤-1,6 branches provide an extra degree of freedom by the rotation about the C-5 and C-6 bonds of the glucose ring (39). The crystal structure of N-␤GRP complexed with laminarihexaoses arranged in a triple helical form has shown that N-␤GRP interacts with ␤-1,3-glucans through six glucose residues, two from each laminarihexaose chain (14). Our chemical cross-linking exper- Ten microliters of plasma from day 2 5th instar larvae of M. sexta was incubated with 10 g of N-␤GRP2 with and without 100 g of laminarin, soluble complex, or insoluble aggregate, in wells of a 96-well plate for 1 h at 25°C.…”

“…For insect ␤GRP, Kanagawa et al (14) suggested from the crystal structure of N-␤GRP complexed with triple helical laminarihexaoses that an unusually large carbohydrate binding surface on N-␤GRP would enhance its affinity to carbohydrate. We propose that N-␤GRP2 may also employ another strategy in which protein-protein interactions become established as a carbohydrate-protein complex is formed.…”

“…Digested peptide fragments were then analyzed using LC-MS/MS, and intra-and intermolecular cross-linked peptides were identified with the software StavroX (24). In the crystal structure of Plodia and Bombyx N-␤GRPs complexed with laminarihexaose (14), protein molecules pack in a side-by-side fashion through electrostatic interactions in a loop that includes Asp 39 . We previously conducted site-directed mutagenesis of the corresponding aspartic acid residue (Asp 45 in Ref.…”

“…These struc-tural studies have included proposed modes for binding of ␤-1,3-glucan by N-␤GRP. The crystal structure of N-␤GRP complexed with laminarihexaose revealed that the carbohydrate-binding site is located on the convex ␤-sheet surface of the domain, with the bound laminarihexaose existing as a triple helical structure (14). However, much remains to be understood about the binding modes of a longer ␤-1,3-glucan that exhibited higher affinity to N-␤GRP (11,12) and the following molecular mechanisms underlying downstream signaling events in the pro-PO activation pathway, including activation of serine protease zymogens.…”

“…The structure of insect N-␤GRP and its carbohydrate-binding mechanism have been studied in recent years. Insect N-␤GRPs have highly conserved amino acid sequences and all their tertiary structures adopt an immunoglobulin-like ␤-sandwich-fold, with two ␤-sheets forming "convex" and "concave" surfaces (11)(12)(13)(14) (supplemental Fig. S1).…”