Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

Abstract

Cellulolytic microorganisms, like Trichoderma reesei or Clostridium thermocellum, frequently have noncatalytic carbohydrate-binding modules (CBMs) associated with secreted or cell surface bound multidomain carbohydrate-active enzymes (CAZymes) like cellulases. Mostly type-A family CBMs are known to promote cellulose deconstruction by increasing the substrate-bound concentration of cognate cellulase catalytic domains. However, due to the interfacial nature of cellulose hydrolysis and the structural heterogeneity of cellulose, it has been challenging to fully understand the role of CBMs on cellulase activity using classical protein-ligand binding assays. Here, we report a single-molecule CAZyme assay for an industrially relevant processive cellulase Cel7A (from T. reesei) to reveal how subtle CBM1 binding differences can drastically impact cellulase motility/velocity and commitment to initial processive motion for deconstruction of two wellstudied crystalline cellulose allomorphs (namely cellulose I and III). We take a multifaceted approach to characterize the complex binding interactions of all major type-A family representative CBMs including CBM1, using an optical-tweezers based single-molecule CBM-cellulose bond 'rupture' assay to complement several classical bulk ensemble protein-ligand binding characterization methods. While our work provides a basis for the 'cautious' use of Langmuir-type adsorption models to characterize classical protein-ligand binding assay data, we highlight the critical limitations of using such overly simplistic models to gain a truly molecular-level understanding of interfacial protein binding interactions at heterogeneous solid-liquid interfaces. Finally, molecular dynamics simulations provided a theoretical basis for the complex binding behavior seen for CBM1 towards two distinct cellulose allomorphs reconciling experimental findings from multiscale analytical methods. Significance StatementMultimodal biomolecular binding interactions involving carbohydrate polymers (e.g., cellulose, starch, chitin, glycosaminoglycans) are fundamental molecular processes relevant to the recognition, biosynthesis, and degradation of all major terrestrial and aquatic biomass. Protein-carbohydrate binding interactions are also critical to industrial biotechnology operations such as enzymatically-catalyzed bioconversion of starch and lignocellulose into biochemicals like ethanol. However, despite the ubiquitous importance of such interfacial processes, we have a poor molecular-level understanding of protein-polysaccharide binding interactions. Here, we provide a comprehensive experimental and theoretical analysis of bulk ensemble versus singlemolecule binding interactions of enzyme motors and associated non-catalytic binding domains with cellulosic polysaccharides to highlight the critical limitations of applying classical biochemical assay techniques alone to understanding protein adsorption or biological activity at solid-liquid interfaces. Main Text

Reduced type‐A carbohydrate‐binding module interactions to cellulose I leads to improved endocellulase activity

“…These binding assay results are summarized in Figure 2D and indicate that native CBMs show ~5 to 12-fold reduction in binding partition coefficient towards cellulose III. This trend is in alignment with our recent study which shows that Type-A CBMs, such as CBM1 (Chundawat et al, 2020), experience major steric clashes with the non-native surface of cellulose III due to the uneven topology of hydrophobic face as shown in Figure 2B that impairs CBM binding. Furthermore, CBM binding reversibility is often overlooked in the literature and this has led to contradictory results being reported (Jervis et al, 1997;Lim et al, 2014;Linder and Teeri, 1996).…”

“…Qualitatively, these mutations seem to impact binding to both cellulose I and cellulose III in a similar manner by reducing binding affinity. It is likely that the loss of hydrophobic stacking interactions between the aromatic residues and cellulose chains impacts binding to both allomorphs (Chundawat et al, 2020;Georgelis et al, 2012). To further understand the impact of these mutations on hydrolytic activity, we produced and characterized the CelE-CBM mutant constructs as described below.…”