Abstract
Cellulosomes are bacterial protein complexes that bind and efficiently degrade lignocellulosic substrates. These are formed by multimodular scaffolding proteins known as scaffoldins, which comprise cohesin modules capable of binding dockerin-bearing enzymes and usually a carbohydrate-binding module that anchors the system to a substrate. It has been suggested that cellulosomes bound to the bacterial cell surface might be exposed to significant mechanical forces. Accordingly, the mechanical properties of these anchored cellulosomes may be important to understand and improve cellulosome function. Here we used single-molecule force spectroscopy to study the mechanical properties of selected cohesin modules from scaffoldins of different cellulosomes. We found that cohesins located in the region connecting the cell and the substrate are more robust than those located outside these two anchoring points. This observation applies to cohesins from primary scaffoldins (i.e. those that directly bind dockerin-bearing enzymes) from different cellulosomes despite their sequence differences. Furthermore, we also found that cohesin nanomechanics (specifically, mechanostability and the position of the mechanical clamp of cohesin) are not significantly affected by other cellulosomal components, including linkers between cohesins, multiple cohesin repeats, and dockerin binding. Finally, we also found that cohesins (from both the connecting and external regions) have poor refolding efficiency but similar refolding rates, suggesting that the high mechanostability of connecting cohesins may be an evolutionarily conserved trait selected to minimize the occurrence of cohesin unfolding, which could irreversibly damage the cellulosome. We conclude that cohesin mechanostability is a major determinant of the overall mechanical stability of the cellulosome.