The O-glycosylated linker from the Trichoderma reesei Family 7 cellulase is a flexible, disordered protein

Abstract

Fungi and bacteria secrete glycoprotein cocktails to deconstruct cellulose. Cellulose-degrading enzymes (cellulases) are often modular, with catalytic domains for cellulose hydrolysis and carbohydrate-binding modules connected by linkers rich in serine and threonine with O-glycosylation. Few studies have probed the role that the linker and O-glycans play in catalysis. Since different expression and growth conditions produce different glycosylation patterns that affect enzyme activity, the structure-function relationships that glycosylation imparts to linkers are relevant for understanding cellulase mechanisms. Here, the linker of the Trichoderma reesei Family 7 cellobiohydrolase (Cel7A) is examined by simulation. Our results suggest that the Cel7A linker is an intrinsically disordered protein with and without glycosylation. Contrary to the predominant view, the O-glycosylation does not change the stiffness of the linker, as measured by the relative fluctuations in the end-to-end distance; rather, it provides a 16 Å extension, thus expanding the operating range of Cel7A. We explain observations from previous biochemical experiments in the light of results obtained here, and compare the Cel7A linker with linkers from other cellulases with sequence-based tools to predict disorder. This preliminary screen indicates that linkers from Family 7 enzymes from other genera and other cellulases within T. reesei may not be as disordered, warranting further study.

Figure 1

Cel7A from Trichoderma reesei on cellulose. The conformation is from Zhong et al. (63) with added N- and O-glycan. The enzyme is shown in gray, the O-glycosylation on the linker is in yellow, and the N-glycan on the CD is in blue. Cellulose is in green, with a single cellodextrin chain threaded into the CD tunnel. The pictures were prepared with VMD (64).

Figure 2

Cel7A linker domain sequence, with the O-glycan shown in red. Adapted from Harrison et al. (11).

Figure 3

Relative free energy as a function of the end-to-end distance (R) from the REMD simulations for both glycosylated and nonglycosylated linkers from T. reesei Cel7A. R₀ for the nonglycosylated linker is 37 Å, and R₀ for the glycosylated linker is 53 Å.

Figure 4

Relative free energy as a function of the mass-weighted R_g for the glycosylated and nonglycosylated T. reesei Cel7A linkers. R_g for the glycosylated linker includes the glycans, and thus the distribution is narrower because the sugars are close to the center of the peptide.

Figure 5

Relative free energy as a function of the end-to-end distance for (a) the putative hinge (R_hinge) and (b) the glycosylated stiff region (R_stiff).