Abstract Resistant starch is a prebiotic with breakdown by gut bacteria requiring the action of specialized amylases and starch-binding proteins. The human gut symbiont Ruminococcus bromii expresses granular starch-binding protein Sas6 (Starch Adherence System member 6) that consists of two starch-specific carbohydrate binding modules from family 26 (RbCBM26) and family 74 (RbCBM74). Here we present the crystal structures of Sas6 and Rb CBM74 with a double helical dimer of maltodecaose bound along an extended surface groove. Binding data combined with native mass spectrometry suggest that RbCBM26 binds short maltooligosaccharides while RbCBM74 can bind single and double helical α-glucans. Our results support a model by which RbCBM74 and RbCBM26 bind neighboring α-glucan chains at the granule surface. CBM74s are conserved among starch granule-degrading bacteria and our work provides molecular insight into how this structure is accommodated by select gut species.

TheRuminococcus bromiiamylosome protein Sas6 binds single and double helical α-glucan structures in starch