Computational investigation of glycosylation effects on a family 1 carbohydrate-binding module

Abstract

Carbohydrate-binding modules (CBMs) are ubiquitous components of glycoside hydrolases, which degrade polysaccharides in nature. CBMs target specific polysaccharides, and CBM binding affinity to cellulose is known to be proportional to cellulase activity, such that increasing binding affinity is an important component of performance improvement. To ascertain the impact of protein and glycan engineering on CBM binding, we use molecular simulation to quantify cellulose binding of a natively glycosylated Family 1 CBM. To validate our approach, we first examine aromatic-carbohydrate interactions on binding, and our predictions are consistent with previous experiments, showing that a tyrosine to tryptophan mutation yields a 2-fold improvement in binding affinity. We then demonstrate that enhanced binding of 3-6-fold over a nonglycosylated CBM is achieved by the addition of a single, native mannose or a mannose dimer, respectively, which has not been considered previously. Furthermore, we show that the addition of a single, artificial glycan on the anterior of the CBM, with the native, posterior glycans also present, can have a dramatic impact on binding affinity in our model, increasing it up to 140-fold relative to the nonglycosylated CBM. These results suggest new directions in protein engineering, in that modifying glycosylation patterns via heterologous expression, manipulation of culture conditions, or introduction of artificial glycosylation sites, can alter CBM binding affinity to carbohydrates and may thus be a general strategy to enhance cellulase performance. Our results also suggest that CBM binding studies should consider the effects of glycosylation on binding and function.

FIGURE 1.

The catalytically active complex of the T. reesei Family 7 cellobiohydrolase (light purple) on cellulose (green) (–19). The native O-glycosylation is shown in yellow and the native N-glycans are shown in dark blue. The CBM is the small protein domain on the left containing 2 native O-glycans, and the catalytic domain is the large protein domain on the right with a cellulose chain threaded into the tunnel.

FIGURE 2.

Side view (A) and top view (B) of the Cel7A CBM and the top layer of the cellulose slab. The tyrosine residues are shown in yellow. The native O-glycans considered here are shown on Thr-1 and Ser-3. The artificial glycan examined here is shown on Ser-14.

FIGURE 3.

Langmuir isotherms of bound CBM as a function of free CBM as measured by Linder et al. (13, 14) (symbols) and predicted by the free energy simulations conducted in this work (lines). Our results indicate improvement in binding affinity over that of just amino acid mutations with the incorporation of a single or dimer O-mannose at Ser-3 (3–6-fold improvement in binding affinity) and a synthetic glycan site at Ser-14 with and without the native glycans (20–140-fold improvement in binding affinity, respectively).

FIGURE 4.

Comparison of the total interaction energy (total energy = electrostatic + van der Waals) between the glycans and cellulose surface present in the S3M1, S14M1-NG, S3M2, and S14M1 100-ns MD simulations. The Thr-1 mannose has zero interaction energy with the surface in each simulation.