Binding preferences, surface attachment, diffusivity, and orientation of a family 1 carbohydrate-binding module on cellulose.

Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 µs of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose.

Modeling the self-assembly of the cellulosome enzyme complex.

Most bacteria use free enzymes to degrade plant cell walls in nature. However, some bacteria have adopted a different strategy wherein enzymes can either be free or tethered on a protein scaffold forming a complex called a cellulosome. The study of the structure and mechanism of these large macromolecular complexes is an active and ongoing research topic, with the goal of finding ways to improve biomass conversion using cellulosomes. Several mechanisms involved in cellulosome formation remain unknown, including how cellulosomal enzymes assemble on the scaffoldin and what governs the population of cellulosomes created during self-assembly. Here, we present a coarse-grained model to study the self-assembly of cellulosomes. The model captures most of the physical characteristics of three cellulosomal enzymes (Cel5B, CelS, and CbhA) and the scaffoldin (CipA) from Clostridium thermocellum. The protein structures are represented by beads connected by restraints to mimic the flexibility and shapes of these proteins. From a large simulation set, the assembly of cellulosomal enzyme complexes is shown to be dominated by their shape and modularity. The multimodular enzyme, CbhA, binds statistically more frequently to the scaffoldin than CelS or Cel5B. The enhanced binding is attributed to the flexible nature and multimodularity of this enzyme, providing a longer residence time around the scaffoldin. The characterization of the factors influencing the cellulosome assembly process may enable new strategies to create designers cellulosomes.

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

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.

The energy landscape for the interaction of the family 1 carbohydrate-binding module and the cellulose surface is altered by hydrolyzed glycosidic bonds.

A multiscale simulation model is used to construct potential and free energy surfaces for the carbohydrate-binding module [CBM] from an industrially important cellulase, Trichoderma reesei cellobiohydrolase I, on the hydrophobic face of a coarse-grained cellulose Ibeta polymorph. We predict from computation that the CBM alone exhibits regions of stability on the hydrophobic face of cellulose every 5 and 10 A, corresponding to a glucose unit and a cellobiose unit, respectively. In addition, we predict a new role for the CBM: specifically, that in the presence of hydrolyzed cellulose chain ends, the CBM exerts a thermodynamic driving force to translate away from the free cellulose chain ends. This suggests that the CBM is not only required for binding to cellulose, as has been known for two decades, but also that it has evolved to both assist the enzyme in recognizing a cellulose chain end and exert a driving force on the enzyme during processive hydrolysis of cellulose.

Molecular modeling suggests induced fit of Family I carbohydrate-binding modules with a broken-chain cellulose surface.

Cellobiohydrolases are the most effective single component of fungal cellulase systems; however, their molecular mode of action on cellulose is not well understood. These enzymes act to detach and hydrolyze cellodextrin chains from crystalline cellulose in a processive manner, and the carbohydrate-binding module (CBM) is thought to play an important role in this process. Understanding the interactions between the CBM and cellulose at the molecular level can assist greatly in formulating selective mutagenesis experiments to confirm the function of the CBM. Computational molecular dynamics was used to investigate the interaction of the CBM from Trichoderma reesei cellobiohydrolase I with a model of the (1,0,0) cellulose surface modified to display a broken chain. Initially, the CBM was located in different positions relative to the reducing end of this break, and during the simulations it appeared to translate freely and randomly across the cellulose surface, which is consistent with its role in processivity. Another important finding is that the reducing end of a cellulose chain appears to induce a conformational change in the CBM. Simulations show that the tyrosine residues on the hydrophobic surface of the CBM, Y5, Y31 and Y32 align with the cellulose chain adjacent to the reducing end and, importantly, that the fourth tyrosine residue in the CBM (Y13) moves from its internal position to form van der Waals interactions with the cellulose surface. As a consequence of this induced change near the surface, the CBM straddles the reducing end of the broken chain. Interestingly, all four aromatic residues are highly conserved in Family I CBM, and thus this recognition mechanism may be universal to this family.

Computer simulation studies of microcrystalline cellulose Ibeta.

Molecular mechanics (MM) simulations have been used to model two small crystals of cellulose Ibeta surrounded by water. These small crystals contained six different extended surfaces: (110), (11 0), two types of (100), and two types of (010). Significant changes took place in the crystal structures. In both crystals there was an expansion of the unit cell, and a change in the gamma angle to almost orthogonal. Both microcrystals developed a right-hand twist of about 1.5 degrees per cellobiose unit, similar to the twisting of beta-sheets in proteins. In addition, in every other layer, made up of the unit cell center chains, a tilt of the sugar rings of 14.8 degrees developed relative to the crystal plane as a result of a transition of the primary alcohol groups in these layers away from the starting TG conformation to GG. In this conformation, these groups made interlayer hydrogen bonds to the origin chains above and below. No change in the primary alcohol conformations or hydrogen-bonding patterns in the origin chain layers was observed. Strong localization of the adjacent water was found for molecules in the first hydration layer of the surfaces, due to both hydrogen bonding to the hydroxyl groups of the sugar molecules and also due to hydrophobic hydration of the extensive regions of nonpolar surface resulting from the axial aliphatic hydrogen atoms of the 'tops' of the glucose monomers. Significant structuring of the water was found to extend far out into the solution. It is hypothesized that the structured layers of water might present a barrier to the approach of cellulase enzymes toward the cellulose surfaces in enzyme-catalyzed hydrolysis, and might inhibit the escape of soluble products, contributing to the slow rates of hydrolysis observed experimentally. Since the water structuring is different for the different surfaces, this might result in slower hydrolysis rates for some surfaces compared to others.

3D electron tomography of pretreated biomass informs atomic modeling of cellulose microfibrils.

Fundamental insights into the macromolecular architecture of plant cell walls will elucidate new structure-property relationships and facilitate optimization of catalytic processes that produce fuels and chemicals from biomass. Here we introduce computational methodology to extract nanoscale geometry of cellulose microfibrils within thermochemically treated biomass directly from electron tomographic data sets. We quantitatively compare the cell wall nanostructure in corn stover following two leading pretreatment strategies: dilute acid with iron sulfate co-catalyst and ammonia fiber expansion (AFEX). Computational analysis of the tomographic data is used to extract mathematical descriptions for longitudinal axes of cellulose microfibrils from which we calculate their nanoscale curvature. These nanostructural measurements are used to inform the construction of atomistic models that exhibit features of cellulose within real, process-relevant biomass. By computational evaluation of these atomic models, we propose relationships between the crystal structure of cellulose Iß and the nanoscale geometry of cellulose microfibrils.

Comparison of Cellulose Iß Simulations with Three Carbohydrate Force Fields.

Molecular dynamics simulations of cellulose have recently become more prevalent due to increased interest in renewable energy applications, and many atomistic and coarse-grained force fields exist that can be applied to cellulose. However, to date no systematic comparison between carbohydrate force fields has been conducted for this important system. To that end, we present a molecular dynamics simulation study of hydrated, 36-chain cellulose Iß microfibrils at room temperature with three carbohydrate force fields (CHARMM35, GLYCAM06, and Gromos 45a4) up to the near-microsecond time scale. Our results indicate that each of these simulated microfibrils diverge from the cellulose Iß crystal structure to varying degrees under the conditions tested. The CHARMM35 and GLYCAM06 force fields eventually result in structures similar to those observed at 500 K with the same force fields, which are consistent with the experimentally observed high-temperature behavior of cellulose I. The third force field, Gromos 45a4, produces behavior significantly different from experiment, from the other two force fields, and from previously reported simulations with this force field using shorter simulation times and constrained periodic boundary conditions. For the GLYCAM06 force field, initial hydrogen-bond conformations and choice of electrostatic scaling factors significantly affect the rate of structural divergence. Our results suggest dramatically different time scales for convergence of properties of interest, which is important in the design of computational studies and comparisons to experimental data. This study highlights that further experimental and theoretical work is required to understand the structure of small diameter cellulose microfibrils typical of plant cellulose.

Harnessing glycosylation to improve cellulase activity.

Cellulases and hemicellulases are responsible for the turnover of plant cell wall polysaccharides in the biosphere, and thus form the foundation of enzyme engineering efforts in biofuels research. Many of these carbohydrate-active enzymes from filamentous fungi contain both N-linked and O-linked glycosylation, the extent and heterogeneity of which depends on growth conditions, expression host, and the presence of glycan trimming enzymes in the secretome, all of which in turn impact enzyme activity. As the roles of glycosylation in enzyme function have not been fully elucidated, here we discuss the potential roles of glycosylation on glycoside hydrolase enzyme structure and function after secretion. We posit that glycosylation, instead of hindering cellulase engineering, can be used as an additional tool to enhance enzyme activity, given deeper understanding of its molecular-level role in biomass deconstruction.

High-temperature behavior of cellulose I.

We use molecular simulation to elucidate the structural behavior of small hydrated cellulose Iß microfibrils heated to 227 °C (500 K) with two carbohydrate force fields. In contrast to the characteristic two-dimensional hydrogen-bonded layer sheets present in the cellulose Iß crystal structure, we show that at high temperature a three-dimensional hydrogen bond network forms, made possible by hydroxymethyl groups changing conformation from trans-gauche (TG) to gauche-gauche (GG) in every second layer corresponding to "center" chains in cellulose Iß and from TG to gauche-trans (GT) in the "origin" layer. The presence of a regular three-dimensional hydrogen bond network between neighboring sheets eliminates the possibility of twist, whereas two-dimensional hydrogen bonding allows for microfibril twist to occur. Structural features of this high-temperature phase as determined by molecular simulation may explain several experimental observations for which no detailed structural basis has been offered. This includes an explanation for the observed temperature and crystal size dependence for the extent of hydrogen/deuterium exchange, and diffraction patterns of cellulose at high temperature.

Coarse-Grain Model for Glucose, Cellobiose, and Cellotetraose in Water.

We present a coarse-grain (CG) simulation model for aqueous solutions of ß-d-glucose, cellobiose, and cellotetraose, based on atomistic simulation data for each system. In the model, three spherical beads are used to represent glucose, and a single bead is used to represent water. For glucose, the force field is calculated using force matching by minimizing the sum of the square differences between forces calculated from atomistic and CG simulations. For cellobiose and cellotetraose, we use a hybrid method where the nonbonded interactions are obtained using force matching and the bonded interactions are obtained using Boltzmann inversion. We demonstrate excellent agreement in the structural properties between the atomistic simulations and the CG simulations. This model represents the first step in developing a CG force field for cellulose, as it is of significant interest to study cellulose behavior at much longer time and length scales relative to atomistic simulations.

Molecular-level origins of biomass recalcitrance: decrystallization free energies for four common cellulose polymorphs.

Cellulose is a crystalline polymer of ß1,4-D-glucose that is difficult to deconstruct to sugars by enzymes. The recalcitrance of cellulose microfibrils is a function of both the shape of cellulose microfibrils and the intrinsic work required to decrystallize individual chains, the latter of which is calculated here from the surfaces of four crystalline cellulose polymorphs: cellulose Iß, cellulose Iα, cellulose II, and cellulose III(I). For edge chains, the order of decrystallization work is as follows (from highest to lowest): Iß, Iα, ΙΙΙ(Ι), and II. For cellulose Iß, we compare chains from three different locations on the surface and find that an increasing number of intralayer hydrogen bonds (from 0 to 2) increases the intrinsic decrystallization work. From these results, we propose a microkinetic model for the deconstruction of cellulose (and chitin) by processive enzymes, which when taken with a previous study [Horn et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 18089] identifies the thermodynamic and kinetic attributes of enzyme and substrate engineering for enhanced cellulose (or chitin) conversion. Overall, this study provides new insights into the molecular interactions that form the structural basis of cellulose, which is the primary building block of plant cell walls, and highlights the need for experimentally determining microfibril shape at the nanometer length scale when comparing conversion rates of cellulose polymorphs by enzymes.

Identification of amino acids responsible for processivity in a Family 1 carbohydrate-binding module from a fungal cellulase.

We probe the molecular-level behavior of the Family 1 carbohydrate-binding module (CBM) from a commonly studied fungal cellulase, the Family 7 cellobiohydrolase (Cel7A) from Trichoderma reesei, on the hydrophobic face of crystalline cellulose. With a fully atomistic model, we predict that the CBM alone exhibits regions of thermodynamic stability along a cellulose chain corresponding to a cellobiose unit, which is the catalytic product of the entire Cel7A enzyme. In addition, we determine which residues and the types of interactions that are responsible for the observed processivity length scale of the CBM: Y5, Q7, N29, and Y32. These results imply that the CBM can anchor the Cel7A enzyme at discrete points along a cellulose chain and thus aid in both recognizing cellulose chain ends for initial attachment to cellulose as well as aid in enzymatic catalysis by diffusing between stable wells on a length scale commensurate with the catalytic, processive cycle of Cel7A during cellulose hydrolysis. Comparison of other Family 1 CBMs show high functional homology to the four amino acids responsible for the processivity length scale on the surface of crystalline cellulose, which suggests that Family 1 CBMs may generally employ this type of approach for translation on the cellulose surface. Overall, this work provides further insight into the molecular-level mechanisms by which a CBM recognizes and interacts with cellulose.

Computational simulations of the Trichoderma reesei cellobiohydrolase I acting on microcrystalline cellulose Ibeta: the enzyme-substrate complex.

Cellobiohydrolases are the dominant components of the commercially relevant Trichoderma reesei cellulase system. Although natural cellulases can totally hydrolyze crystalline cellulose to soluble sugars, the current enzyme loadings and long digestion times required render these enzymes less than cost effective for biomass conversion processes. It is clear that cellobiohydrolases must be improved via protein engineering to reduce processing costs. To better understand cellobiohydrolase function, new simulations have been conducted using charmm of cellobiohydrolase I (CBH I) from T.reesei interacting with a model segment (cellodextrin) of a cellulose microfibril in which one chain from the substrate has been placed into the active site tunnel mimicking the hypothesized configuration prior to final substrate docking (i.e., the +1 and +2 sites are unoccupied), which is also the structure following a catalytic bond scission. No tendency was found for the protein to dissociate from or translate along the substrate surface during this initial simulation, nor to align with the direction of the cellulose chains. However, a tendency for the decrystallized cellodextrin to partially re-anneal into the cellulose surface hints that the arbitrary starting configuration selected was not ideal.