Structural insights into xyloglucan recognition by an ABC transporter from a Gram‐positive, thermophilic bacterium

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. To our knowledge, Athe_2052 is the first structurally characterized ABC SBP known to recognize 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 n

) 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.