Mapping Molecular Recognition of β1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus

A multigene polysaccharide utilization locus (PUL) encoding enzymes and surface carbohydrate (glycan)-binding proteins (SGBPs) was recently identified in prominent members of Bacteroidetes in the human gut and characterized in Bacteroides ovatus. This PUL-encoded system specifically targets mixed-linkage beta 1,3-1,4-glucans, a group of diet-derived carbohydrates that promote a healthy microbiota and have potential as prebiotics. The BoSGBP(MLG)-A protein encoded by the BACOVA_2743 gene is a SusD-like protein that plays a key role in the PUL's specificity and functionality. Here, we perform a detailed analysis of the molecular determinants underlying carbohydrate binding by BoSGBP(MLG)-A, combining carbohydrate microarray technology with quantitative affinity studies and a high-resolution X-ray crystallography structure of the complex of BoSGBP(MLG)-A with a beta 1,3-1,4-nonasaccharide. We demonstrate its unique binding specificity toward beta 1,3-1,4-gluco-oligosaccharides, with increasing binding affinities up to the octasaccharide and dependency on the number and position of beta 1,3 linkages. The interaction is defined by a 41-angstrom-long extended binding site that accommodates the oligosaccharide in a mode distinct from that of previously described bacterial beta 1,3-1,4-glucan-binding proteins. In addition to the shape complementarity mediated by CH-pi interactions, a complex hydrogen bonding network complemented by a high number of key ordered water molecules establishes additional specific interactions with the oligosaccharide. These support the twisted conformation of the beta-glucan backbone imposed by the beta 1,3 linkages and explain the dependency on the oligosaccharide chain length. We propose that the specificity of the PUL conferred by BoSGBP(MLG)-A to import long beta 1,3-1,4-glucan oligosaccharides to the bacterial periplasm allows Bacteroidetes to outcompete bacteria that lack this PUL for utilization of beta 1,3-1,4-glucans. IMPORTANCE With the knowledge of bacterial gene systems encoding proteins that target dietary carbohydrates as a source of nutrients and their importance for human health, major efforts are being made to understand carbohydrate recognition by various commensal bacteria. Here, we describe an integrative strategy that combines carbohydrate microarray technology with structural studies to further elucidate the molecular determinants of carbohydrate recognition by BoSGBP(MLG)-A, a key protein expressed at the surface of Bacteroides ovatus for utilization of mixed-linkage beta 1,3-1,4-glucans. We have mapped at high resolution interactions that occur at the binding site of BoSGBP(MLG)-A and provide evidence for the role of key water-mediated interactions for fine specificity and affinity. Understanding at the molecular level how commensal bacteria, such as prominent members of Bacteroidetes, can differentially utilize dietary carbohydrates with potential prebiotic activities will shed light on possible ways to modulate the microbiome to promote human health.

Automated summary

A multi-gene polysaccharide utilization locus (PUL) targeting mixed-linkage beta 1,3-1,4-glucans has been identified in prominent members of Bacteroidetes in the human gut, which plays a crucial role in promoting a healthy microbiota and as potential prebiotics. The BoSGBP(MLG)-A protein shows unique binding specificity towards beta 1,3-1,4-gluco-oligosaccharides, with interactions characterized by a complex hydrogen bonding network and specific interactions with ordered water molecules. Understanding the molecular mechanisms of carbohydrate recognition by commensal bacteria like Bacteroidetes can provide insights into modulating the microbiome for promoting human health.