Structural and Biochemical Characterization of a Nonbinding SusD-Like Protein Involved in Xylooligosaccharide Utilization by an Uncultured Human Gut <i>Bacteroides</i> Strain.

In the human gut microbiota, <i>Bacteroidetes</i> break down dietary and endogenous glycosides through highly specific polysaccharide utilization loci (PULs). PULs encode a variety of sensor regulators, binding proteins, transporters, and carbohydrate-active enzymes (CAZymes). Surface glycan-binding proteins (SGBPs) are essential for the efficient capture of the glycosides present on the cell surface, providing <i>Bacteroidetes</i> with a competitive advantage in colonizing their habitats. Here, we present the functional and structural characterization of a SusD-like protein encoded by a xylooligosaccharide (XOS) PUL from an uncultured human gut <i>Bacteroides</i> strain. This locus is also conserved in Bacteroides vulgatus, thereby providing new mechanistic insights into the role of SGBPs in the metabolism of dietary fiber of importance for gut health. Various <i>in vitro</i> analyses, including saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, revealed that the SusD-like protein cannot bind to the cognate substrate of the XOS PUL, although its presence is essential for the PUL to function. Analysis of the crystal structure of the SusD-like protein reveals an unfolded binding surface and the absence or inappropriate orientation of several key residues compared with other known SusD-like structures. These results highlight the critical role of the SusD-like protein in the transport of oligosaccharides and provide fundamental knowledge about the structure-function of SusC/D-like transporters, revealing that the binding specificity of SusD-like SGBPs does not necessarily reflect the uptake specificity of the transporter. <b>IMPORTANCE</b> The metabolization of dietary fiber is a crucial function for many gut bacteria, especially <i>Bacteroidetes</i>, which are particularly well adapted for recognizing, binding, transporting, and degrading glycosides. In this study, we report the functional and structural characterization of a SusD-like protein involved in xylooligosaccharide utilization by an uncultured gut <i>Bacteroides</i> strain. We demonstrate that while this protein is structurally similar to many canonical <i>Bacteroidetes</i> surface glycan-binding proteins, it cannot bind the substrate taken up by the cognate SusC-like transporter. This lack of binding might be explained by the absence of several key residues known to be involved in oligosaccharide binding and/or the possible necessity of the SusC-like protein to be present to create a cooperative binding site. The term "surface glycan-binding proteins" generally used for SusD-like proteins is thus not generic. Overall, this study allowed us to revisit the concept of glycoside utilization by <i>Bacteroidetes</i>, in particular those strains that feed on the short fibers naturally present in some dietary compounds or on the leftovers of other microbes.