Abstract
Xyloglucan (an α-1,6-xylosyl-substituted β-1,4-glucan) is a major hemicellulose of the primary cell wall of many plants and an important growth substrate for biomass-degrading bacteria in diverse ecological niches, including the gut microbiome and hot springs. In Gram-positive bacteria, xyloglucan is deconstructed into soluble oligosaccharides in the extracytoplasmic space before import by ATP-Binding Cassette (ABC) transporters, but the structural basis for this process remains poorly understood. Here, we identified an ABC transporter for xyloglucan uptake (Athe_2052-2054) in the Gram-positive, plant biomass-degrading thermophile Anaerocellum bescii, which is conserved across the Anaerocellum genus. We solved the apo crystal structure of its extracellular substrate-binding protein (SBP), Athe_2052, revealing a unique tertiary fold found only in a small subset of SBPs that bind complex oligosaccharides. This structure represents the first ABC SBP known to bind xyloglucan oligosaccharides. Biophysical analysis showed that while Athe_2052 binds unsubstituted β-glucan chains, recognition of xyloglucan side chains in the binding pocket markedly increases affinity (K(d) = 14 nM) for xyloglucan heptasaccharide (XXXG), the principal oligosaccharide released during xyloglucan deconstruction. Molecular modeling revealed that xyloglucan heptasaccharide, owing to its branched substitutions, is bound in a distinct conformation compared to unsubstituted β-glucans. This represents a unique mode of xyloglucan recognition driven by α-linked side-chain interactions rather than β-glucan backbone recognition alone. Together, these findings provide the first structural basis for xyloglucan oligosaccharide recognition by an ABC transporter in Gram-positive bacteria.