Molecular origins of reduced activity and binding commitment of processive cellulases and associated carbohydrate-binding proteins to cellulose III

Shishir P.S Chundawat,Bhargava Nemmaru,Markus Hackl,Sonia K Brady,Mark A Hilton,Madeline M Johnson,Sungrok Chang,Matthew J Lang,Hyun Huh,Sang-Hyuk Lee,John M Yarbrough,Cesar A López,Sandrasegaram Gnanakaran

doi:10.1016/j.jbc.2021.100431

Abstract

Efficient enzymatic saccharification of cellulosic biomass into fermentable sugars can enable production of bioproducts like ethanol. Native crystalline cellulose, or cellulose I, is inefficiently processed via enzymatic hydrolysis but can be converted into the structurally distinct cellulose III allomorph that is processed via cellulase cocktails derived from Trichoderma reesei up to 20-fold faster. However, characterization of individual cellulases from T. reesei, like the processive exocellulase Cel7A, shows reduced binding and activity at low enzyme loadings toward cellulose III. To clarify this discrepancy, we monitored the single-molecule initial binding commitment and subsequent processive motility of Cel7A enzymes and associated carbohydrate-binding modules (CBMs) on cellulose using optical tweezers force spectroscopy. We confirmed a 48% lower initial binding commitment and 32% slower processive motility of Cel7A on cellulose III, which we hypothesized derives from reduced binding affinity of the Cel7A binding domain CBM1. Classical CBM–cellulose pull-down assays, depending on the adsorption model fitted, predicted between 1.2- and 7-fold reduction in CBM1 binding affinity for cellulose III. Force spectroscopy measurements of CBM1–cellulose interactions, along with molecular dynamics simulations, indicated that previous interpretations of classical binding assay results using multisite adsorption models may have complicated analysis, and instead suggest simpler single-site models should be used. These findings were corroborated by binding analysis of other type-A CBMs (CBM2a, CBM3a, CBM5, CBM10, and CBM64) on both cellulose allomorphs. Finally, we discuss how complementary analytical tools are critical to gain insight into the complex mechanisms of insoluble polysaccharides hydrolysis by cellulolytic enzymes and associated carbohydrate-binding proteins.

Highlights

Thermochemical pretreatment using acids, bases, or ionic liquids is employed to increase polysaccharide accessibility to enzymes and reduce nonproductive cellulase binding [9,10,11]
Cel7A catalytic domain (CD) in the absence of CBM1 showed lower dwell times between catalytic turnover steps suggesting that carbohydrate-binding module (CBM) could impede full-length cellulase motility on native cellulose I owing to nonproductive binding
Spectroscopic characterization using X-ray diffraction (XRD) and Fourier transform Raman spectroscopy were conducted to confirm the conversion of cellulose I to cellulose III allomorph following ammonia pretreatment and measure substrate characteristics like cellulose crystallinity index (CrI) and crystallite size

Summary

Introduction

Thermochemical pretreatment using acids, bases, or ionic liquids is employed to increase polysaccharide accessibility to enzymes and reduce nonproductive cellulase binding [9,10,11]. Single-molecule fluorescence imaging allows estimation of exocellulase binding kinetics parameters (e.g., adsorption and desorption rates) [8, 22, 23], whereas high-speed atomic force microscopy allows tracking of motility of single cellulase molecules [24, 25] These methods cannot resolve the slower subnanometer translational rates of processive cellulases relevant to cellulose decrystallization and hydrolysis into cellobiose. We developed a new optical tweezers–based CBM–cellulose bond “rupture” assay to characterize the binding behavior of single CBM1 proteins alone to distinct cellulose allomorph surfaces under applied force. To generalize these findings further, we characterized CBM3a (another type A CBM from C. thermocellum) using equilibrium pull-down and kinetic binding assays. Our work highlights the necessity of using a multifaceted approach for characterizing the binding heterogeneity and multimodal nature of cellulase–cellulose interactions

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