The Carbohydrate Binding Module of TrCel7A Aids in Navigating the Complexity of Plant Cell Walls

Efficient enzymatic deconstruction of plant cell walls is critical for utilization of lignocellulose biomass. Key enzymes in this process are cellobiohydrolases, a class of cellulases that processively degrade crystalline cellulose. Many cellobiohydrolases possess a carbohydrate-binding module (CBM), yet the importance of CBMs in substrate interaction remains unclear. Here, we use single-molecule fluorescence microscopy to investigate how CBM1 of Trichoderma reesei Cel7A influences enzyme binding and motility on cellulose substrates of varying complexity. We compare wild-type Cel7A with a truncated variant lacking CBM1 (Cel7A{Delta}CBM) on bacterial cellulose (BC), phosphoric acid swollen cellulose (PASC), delignified milkweed cellulose (MWC), and holocellulose nanofibrils (hCNF). While both variants showed similar steady-state binding densities on BC and PASC, Cel7A{Delta}CBM exhibited reduced binding on MWC and hCNF, with the greatest reduction on the hemicellulose-rich hCNF. Alkali removal of hemicellulose partially restored Cel7A{Delta}CBM binding, suggesting a role for CBM1 in substrate navigation and productive binding sites recognition. Kinetic analyses revealed that CBM1 enables a rapid binding mode absent in the truncated variant. Comparisons with isolated CBM3 further showed that CBMs are capable of fast substrate association. These findings demonstrate that CBMs enhance cellulase-substrate interactions by accelerating binding, enabling navigation of the complex environment of plant cell walls. Our results emphasize the importance of CBMs in natural cellobiohydrolase function and highlight their value in the design of improved cellulases for industrial biomass conversion.