The Ruminococcus bromii amylosome protein Sas6 binds single and double helical α-glucan structures in starch

Area of Science:

• Microbiology and Structural Biology
• Gut Microbiome and Host-Microbe Interactions

Background:

• Resistant starch serves as a prebiotic, requiring specialized bacterial enzymes and starch-binding proteins for digestion in the gut.
• The gut symbiont Ruminococcus bromii utilizes Sas6 (Starch Adherence System member 6), a protein comprising two starch-specific carbohydrate-binding modules (RbCBM26 and RbCBM74).

Purpose of the Study:

• To elucidate the molecular mechanisms by which Ruminococcus bromii's Sas6 protein interacts with resistant starch.
• To determine the structural basis for the differential binding of RbCBM74 and RbCBM26 modules to various starch structures.

Main Methods:

• Crystal structure determination of the Sas6 protein and the RbCBM74 module bound to maltodecaose.
• Isothermal titration calorimetry and native mass spectrometry to assess binding affinities and specificities.
• Bioinformatic analysis to investigate the conservation of starch-binding modules in related bacteria.

Main Results:

• The crystal structure of RbCBM74 reveals a binding groove complementary to the double helical structure of amylopectin, suggesting a role in targeting starch granules.
• RbCBM74 preferentially binds longer single and double helical alpha-glucans, whereas RbCBM26 binds shorter maltooligosaccharides.
• Bioinformatic analysis indicates that the amylopectin-targeting platform in CBM74 modules is conserved among bacteria that degrade resistant starch.

Conclusions:

• The Sas6 protein, through its distinct RbCBM74 and RbCBM26 modules, recognizes specific features of the starch granule.
• These findings provide molecular insights into how gut bacteria, like Ruminococcus bromii, effectively utilize resistant starch as a nutrient source.
• The structural and binding data highlight the specialized adaptations of gut symbionts for accessing complex carbohydrates in the human gut.