Engineering protein-carbohydrate binding affinity via glycosylation: A general strategy to improve cellulase performance

Carbohydrate-binding modules (CBMs) display specificity to mono- and polysaccharides for docking of enzyme complexes to carbohydrates. CBMs are found ubiquitously as components of cellulase and hemicellulase enzymes that degrade cell wall polysaccharides in nature. For this purpose, CBMs have evolved to recognize specific cell wall polysaccharides via hydrophobic interactions and hydrogen bonding. Additionally, CBM binding affinity to carbohydrates has been shown to be proportional to enzyme activity, and thus increasing binding affinity is an important component of engineering improved cellulases. To that end, here we apply molecular simulation and free energy calculations to examine the binding affinity of a well-characterized Family 1 CBM to cellulose. To validate our approach, we quantify the impact of aromatic-carbohydrate interactions on the CBM binding affinity. Our predictions are in excellent quantitative agreement with previous experimental results showing that single-point mutations can alter the CBM binding affinity by approximately 2-fold. Following this validation, we demonstrate that a single, native O-linked mannose increases the CBM binding affinity to cellulose by 40-fold, which has not been considered explicitly in previous work. Furthermore, we show that the addition of a single artificial glycan on the CBM can have the same order of magnitude impact on binding affinity. These results have significant implications for protein engineering, in that modifying glycosylation patterns, for example by using heterologous host expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can drastically alter protein binding affinity to carbohydrate substrates, and may thus be a general strategy to improve cellulase performance.