Effects of Xylan Side-Chain Substitutions on XylanâCellulose

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Effects of Xylan Side Chain Substitutions on Xylan-Cellulose Interactions and Implications for Thermal Pretreatment of Cellulosic Biomass Caroline S. Pereira, Rodrigo L. Silveira, Paul Dupree, and Munir S. Skaf Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00067 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017

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Effects of Xylan Side Chain Substitutions on Xylan-Cellulose Interactions and Implications for Thermal Pretreatment of Cellulosic Biomass

Caroline S. Pereira†#, Rodrigo L. Silveira†#, Paul Dupree‡, Munir S. Skaf†*

†

‡

Institute of Chemistry, University of Campinas, Campinas, Sao Paulo, 13084-862, Brazil Department of Biochemistry and the Leverhulme Natural Material Innovation Centre,

University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, United Kingdom # Equally contributing authors. *Corresponding author. E-mail: [email protected]

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Abstract

Lignocellulosic biomass is mainly constituted by cellulose, hemicellulose and lignin and represents an important resource for the sustainable production of biofuels and green chemistry materials. Xylans, a common hemicellulose, interact with cellulose and often exhibit various side chain substitutions including with acetate, (4-O-methyl) glucuronic acid, and arabinose. Recent studies have shown that the distribution of xylan substitutions is not random, but follows patterns that are dependent on the plant taxonomic family and cell wall type. Here, we use molecular dynamics simulations to investigate the role of substitutions on xylan interactions with the hydrophilic cellulose face, using the recently discovered xylan decoration pattern of the conifer gymnosperms as a model. The results show that α-1,2-linked substitutions stabilize the binding of single xylan chains independently of the nature of the substitution and that Ca2+ ions can mediate cross links between glucuronic acid substitutions of two neighboring xylan chains, thus stabilizing binding. At high temperature, xylans move from the hydrophilic to the hydrophobic cellulose surface and are also stabilized by Ca2+ cross links. Our results help to explain the role of substitutions on xylan-cellulose interactions, and improve our understanding of the plant cell wall architecture and the fundamentals of biomass pretreatments.

Keywords: hemicellulose; plant cell wall; pretreatment; lignocellulosic biomass; calcium; molecular dynamics


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Introduction Lignocellulosic biomass constitutes renewable feedstocks that can be converted into biofuels or chemical building blocks for a variety of materials in a sustainable way.1,2 Such biomass is present in the walls of plant cells, which is mostly comprised of cellulose, hemicellulose, and lignin. Despite being extremely abundant in nature, processes currently employed to deconstruct plant cell walls are slow and expensive, thus creating a challenge for their implementation in industrial settings.3 The plant cell wall is a complex network of biopolymers that provides protection to plant cells. The structural and mechanical properties of plant cell walls, as well as their responses to thermochemical treatments, depend on how hemicellulose and lignin interact with the cellulose microfibrils. Understanding hemicelluloses at a molecular level and their interactions with other constituents of plant cell walls has gained considerable attention in recent years since these polysaccharides are essential for plant development and contribute to the biomass recalcitrance against chemical and enzymatic transformation by limiting cellulose accessibility.4 A common practice is to employ thermochemical pretreatments, such as hot water, steam explosion and dilute acid, to remove hemicellulose.3 Thus, understanding molecular aspects of the interactions involving hemicelluloses in plant cell walls would help the development of more cost-effective sustainable technologies, including modulation of hemicellulose composition through plant cell wall engineering.5,6 Cellulose microfibrils in plants are composed of long β-1,4-linked glucan chains. Estimates of the number of glucan chains in each microfibril vary between plant species and by measurement method. For many years it was thought that there are 36 chains in each fibril, but more recently several approaches have led to the belief there are likely 18.7-10 The glucan chains


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are mainly arranged in a Iβ allomorph, but the cross-sectional arrangement is as yet unclear. Square and hexagonal fibril cross sections have been proposed, each presenting differing hydrophilic and hydrophobic faces for interaction with the other cell wall components.11 Such complexity of cellulose fibril models complicates the understanding of these interactions. Xylans are the most abundant type of hemicellulose in grass cell walls, in eudicot secondary cell walls, and a significant part of conifer cell walls, consisting of a main chain of β1,4-linked xylosyl units, often substituted with 2- and 3-linked acetyl, α-1,3-L-arabinofuranosyl (Ara) or α-1,2-(4-O-methyl-)glucuronosyl (MeGlcA) residues.12 In some grass endosperm walls, α-1,2-Ara and α-1,3-Ara disubstituted residues are present. Although the chemical compositions of xylans are relatively well characterized, the extraordinarily precise patterned localization of substitutions has only recently been elucidated.13 For example, we have recently shown that, in conifer gymnosperms, MeGlcA substitutions occur precisely at every sixth xylosyl residue, whereas Ara substitutions occur precisely two residues apart from MeGlcA-substituted xylosyl residues.14 A similar even substitution pattern is found in Arabidopsis,15 but with acetyl branches instead of Ara substitutions. The main feature of these specific substitution patterns is that one of the faces of the xylan chain remains totally unsubstituted and available to interact with the cellulose hydrophilic face through hydrogen bonds in a twofold screw helical conformation, as our recent molecular dynamics (MD) simulations have suggested.14,15 The binding of xylan to cellulose fibrils in cell walls in the twofold screw conformation according to our models has also recently been confirmed by solid state NMR.16 However, any role of the different type and linkage of substitutions in the xylan-cellulose interactions remains poorly understood. The study of hemicellulose-cellulose interactions is not a simple task because of the large spectrum of possible side chain substitutions of hemicellulose and the lack of experimentally


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well-characterized models of cellulose microfibrils regarding shape, size, solvent-exposed surfaces and twist along the main axis. In this study, we performed a large set of MD simulations (a total of 3.6 µs) to elucidate the functions of arabinose and glucuronic acid substitutions in the interactions of xylan with the (110) cellulose hydrophilic face, which is an exposed face in several models of cellulose microfibrils.11 We have simulated complexes of single and multiple xylan chains on cellulose under both ambient and hydrothermal pretreatment thermodynamic conditions. Our choice for type and linkage position of side chain substitutions are restricted to the experimentally determined xylan decoration pattern in gymnosperms recently discovered.13,14 The results show that the binding stability of a single xylan chain can be enhanced by α-1,2linked substitutions independent of the type of substitution. Also, glucuronic acid interactions with Ca2+ ions stabilize the binding of multiple xylan chains on cellulose and, under pretreatment conditions, xylans leave the hydrophilic face to tightly bind the hydrophobic face while crosslinked to one another by glucuronic acid-Ca2+ interactions. The study helps to understand the fundamentals of the plant cell wall architecture as well as its modification by thermochemical processes.

Methods The initial structure of the cellulose microfibril was obtained using Cellulose-Builder,17 considering the Iβ polymorph18 and degree of polymerization (DP) of 22 glucose units. An 18chain model obtained by considering half of the hexagonal 36-chain model9,19 was employed (Fig. 1). This model has both the (110) hydrophilic and (100) hydrophobic faces exposed. A glucan chain of DP 14 was then placed on the hydrophilic surface according to the crystalline arrangement. The hydroxymethyl groups on this chain were removed to generate a xylan


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segment. Due to the lack of publicly available potential parameters for the 4-O-Me group, our model replaces the methoxy by 4-OH in the glucuronic acid substitutions, thus generating α-1,2glucuronopyranosyl decorations (henceforth named, GlcA). Our simulations indicate that the 4OH group of GlcA interacts mostly with the solvent and is only marginally involved in interactions with the polysaccharides (see Supporting Information, Fig. S1). α-1,2-glucuronopyranosyl (GlcA) and α-1,3-L-Arabinofuranosyl (Ara) substitutions were inserted on this xylan chain according to the conifer substitution pattern (Fig. 1) obtained experimentally9 using CHARMM36 internal coordinates.20,21 Systems containing two and three xylan chains were built following the same procedure. The systems were immersed in a box of water 16 Å thick from the xylan-cellulose complex. One calcium ion was added for every two GlcA substitutions present in the system. Effects of ionic strength were evaluated by adding calcium and chloride ions, as described in the Results section. It is important to point out that the starting xylan-cellulose binding motif adopted here, where the chains are initially elongated in an essentially crystalline orientation, is just one of many putative binding conformations. A full exploration of the many binding possibilities is beyond the scope of this work. On that note, it is also important to emphasize that there are compelling experimental evidence that the decoration pattern is not random. Moreover, the starting configuration for systems containing two and three GAX is such that the decorations are in register with each other. We have chosen this particular configuration because it seemed to us the least favorable to binding due the side chain volumes. Many other off-register configurations are possible but have not been investigated here. The simulations were performed employing periodic boundary conditions, with electrostatics handled via particle mesh Ewald22 and short range interactions truncated at a cutoff radius of 12 Å. Chemical bonds involving hydrogen atoms were constrained to their equilibrium


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lengths and a time step of 2 fs was used to solve the equations of motion. Simulations at ambient conditions were run at 300 K and 1 bar, while simulations at high temperature were run at 433 K (160 °C) under constant volume corresponding to the ambient liquid water density. Temperature and pressure were controlled with the Langevin thermostat and piston, respectively.23 To prevent distortions of the cellulose fibril, the C1, C2, C3, C4, C5, and O5 atoms of the cellulose glucose rings were harmonically restrained with a force constant of 50 kcal mol-1 Å-2. All hydroxymethyl groups (C6) were allowed to move freely. The following procedure was employed to prepare the systems for the production runs: (1) 100 steps of energy minimization followed by 500 ps of MD with only the solvent and xylan decoration atoms free; (2) same as (1), but with only cellulose atoms restrained as described above. For each system, sets of three independent simulations lasting 200 ns or 500 ns were generated using NAMD.23 Interactions were modeled with the CHARMM36 force field for carbohydrates20,21 and the TIP3P water model.24 As in any classical MD simulation, the results may be more or less dependent on the choice of the force field and properties of interest. Some finer-grain details of cellulose conformation and the interactions with water obtained from density functional theory quantum calculations25-27 are not captured by classical MD techniques. Analyses were performed with in-house codes, readily available upon request to the authors, and VMD.28

Results Interactions between cellulose and a single xylan chain α-1,3-Ara and α-1,2-GlcA substitutions influence xylan-cellulose interactions. To understand the effects of α-1,3-Ara and α-1,2-GlcA substitutions on xylan-cellulose interactions, we first


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characterized the behavior of a single glucuronoarabinoxylan (GAX) on the (110) hydrophilic surface of cellulose. We employed the same xylan-cellulose complex that we used in our previous study,14 in which GAX exhibits the conifer substitution pattern. In this model, the decorated xylan chain has DP 14 and the following substitution pattern: α-1,3-Ara(3), α-1,2GlcA(5), α-1,3-Ara(9) and α-1,2-GlcA(11), where the numbers in parentheses indicate the xylosyl unit along the xylan backbone (Fig. 1A). Figure 2A shows the global mobility of the GAX chain in three independent simulations, as measured by its root mean squared deviation (rmsd) from the initial stretched conformation (Fig. 1B and 1C). After approximately 50 ns from the start of the simulations, the rmsds fluctuate only locally about constant values, indicating that the GAX chain stably binds to cellulose hydrophilic face and exhibits only minor structural fluctuations while bound. Higher fluctuations observed during the first 50 ns are due to the flexible extremities of the xylan chain. The percentage of xylosyl residues that are bound to the cellulose fibril as a function of time (Fig. 2B) provides a measure of the strength of xylan-cellulose interactions. A given residue was counted as bound when the distance of any atom of xylan to any atom of cellulose was ≤ 2.5 Å (i.e., roughly the typical distance between heavy atoms engaged in hydrogen bonding). The percentage of bound xylosyl residues fluctuates mainly between ~50% and 100%, with an average of ~80% of bound residues. This indicates that, due to thermal fluctuations, some xylosyl residues can locally unbind the cellulose surface, but the GAX chain remains effectively bound to the cellulose surface most of the simulation time. The number of glucan chains of cellulose surface that contact the bound xylan backbone (Fig. 2C) indicates how stretched the GAX chain is on the surface. If the xylan chain is completely stretched and aligned with a single glucan chain, the xylan backbone contacts two


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glucan chains. This can be seen in Fig. 1B, where the GAX contacts both the glucan chain that establishes hydrogen bonds with the xylan backbone and the glucan chain right below it, with which the xylan backbone interacts via carbohydrate stacking. So, the greater the number of glucan chains in contact with the xylan, the more the xylan chain deviates from the totally stretched configuration. Figure 2C shows that GAX contacts mostly between two and four cellulose chains while bound, indicating that the xylan conformation dynamically fluctuates from the fully stretched conformation on the cellulose surface. However, the GAX chain does not assume entirely random configurations over the cellulose surface, as the maximum number of contacted glucan chains is 4 (the brief occurrence of 5 contacts, shown in Fig. 2C, happens only occasionally during the course of the simulations and does not seem to be significant). To assess the effects of α-1,3-Ara and α-1,2-GlcA substitutions on the GAX-cellulose interactions, we performed additional simulations of a single unsubstituted xylan (UX) chain bound to the cellulose surface. The simulations show that UX is less stable than GAX on the (110) face. The rmsd reach values up to 25 Å and are not stabilized around constant values, indicating high mobility of the UX chain (Fig. 2D). The percentage of bound UX xylosyl residues fluctuates between ~20% and 100%, with an average of 65%, indicating weaker binding to the (110) cellulose surface compared to GAX (Fig. 2E). Moreover, UX contacts between 1 and 6 glucan chains on the cellulose fibril, including a chain belonging to the (100) hydrophobic surface (Fig. 2F). These results indicate the prevalence of non-stretched configurations for UX, as opposed to the decorated xylan, and suggest that α-1,3-Ara and/or α-1,2-GlcA substitutions play roles in stabilizing and aligning the xylan chain on the hydrophilic cellulose surface. The behavior of xylans on the cellulose surface is in sharp contrast with that of a glucan chain bound to cellulose, which we also simulated. As shown in Fig. 2G, 2H and 2I, the rmsd of


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the glucan chain remains mostly below 2 Å, the percentage of bound glucosyl residues remains always above 80%, and the number of contacted glucan chains in the cellulose surface varies between 2 and 3. Thus, the lack of C6 hydroxymethyl groups in the xylan chain is responsible for its loose binding to the cellulose surface when compared to a glucan chain, and the substitutions present in GAX are partially able to compensate for their absence.

Stabilizing effects come from α-1,2-GlcA rather than α-1,3-Ara. In order to understand the function played by the substitutions individually, we built and simulated two additional xylan systems on cellulose: arabinoxylan (AX), with four α-1,3-Ara substitutions [α-1,3-Ara(3), α-1,3Ara(5), α-1,3-Ara(9) and α-1,3-Ara(11)], and glucuronoxylan (GX), with four α-1,2-GlcA substitutions [α-1,2-GlcA(3), α-1,2-GlcA(5), α-1,2-GlcA(9) and α-1,2-GlcA(11)]. Strikingly, the behavior of the bound AX resembles that of UX, whereas GX is even more stable than GAX and resembles the glucan chain over the cellulose surface (Fig. 2 and 3). Together, these results indicate that, in GAX, the α-1,2-GlcA substitutions, and not α-1,3-Ara, effectively contribute to stabilize the binding of xylan onto cellulose. Two possibilities may account for this effect: (1) Interactions between cellulose and the α-1,2-GlcA decoration may be stronger than with α-1,3Ara; and/or (2) the position of substitutions in the xylosyl residue matters, e.g., α-1,2- and α-1,3linked substitutions may lead to different effects. Below, we scrutinize the likelihood of these two hypotheses.

Effects of the substitutions arise from the position of the link to the xylan backbone. Closer inspection of the simulations show that, in GAX, AX and GX, the GlcA and Ara substitutions are mobile and able to dynamically contact the cellulose hydrophilic edge even when the xylan backbone is bound to the surface. Results for the number of contacts between the four xylan 10 ACS Paragon Plus Environment

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substitutions and cellulose as function of simulation time have been obtained (Fig. S2) which indicate that AX’s α-1,3-Ara substitutions establish more contacts with cellulose than GX’s α1,2-GlcA. Yet, GX is significantly more tightly bound to cellulose (Fig. 3). Therefore, side chain decoration-cellulose interactions are not the primary reason why α-1,2-GlcA substitutions stabilize the binding of GAX. To assess the effect of the position of the substitutions in the xylosyl residues, we built two additional xylans: AX with α-1,2-Ara substitutions and GX with α-1,3-GlcA substitutions. In marked contrast with the regular α-1,3-Ara and α-1,2-GlcA decorations, α-1,2-Ara substitutions stabilize AX binding, whereas α-1,3-GlcA substitutions do not exhibit significant stabilizing effects when compared to the undecorated xylan UX (Fig. 4A-F). These results indicate that what determines the behavior of xylan decorated with Ara and GlcA on the cellulose surface is mainly the position of the substitution linkages to the xylan backbone, and not the chemical nature of the substitutions. We attribute this behavior to changes in the flexibility of the xylan backbone due to the presence of side chain substitutions (see Fig. S3 for a quantitative assessment of the conformational fluctuations of GAX and UX). On the one hand, the nearest glycosidic bonds to the α-1,3-linked substitutions point towards the cellulose surface (Fig. 5A) and barely interact with the substitutions. Thus, the chemical environment of such glycosidic bonds resembles that of UX. On the other hand, the closest glycosidic bonds to the α-1,2-linked substitutions point away from the cellulose surface and sterically interact with the substitutions (Fig. 5A). Figure 5B shows the distribution of van der Waals (vdW) interactions between the glycosidic oxygens and the α-1,2-GlcA and α-1,3-Ara substitutions in GAX. The data shows that vdW interactions between α-1,2-GlcA substitutions and the glycosidic bonds are mostly repulsive, indicating the


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presence of steric effects, whereas vdW interactions between α-1,3-Ara substitutions and the glycosidic bonds are only slightly attractive, indicating little proximity between the moieties and absence of steric effects. (The vdW energy distribution for α-1,2-Ara, shown in Fig. S4, is similar to that of α-1,2-GlcA; the α-1,3-GlcA energy distribution was not computed because the molecule did not remain bound to cellulose). Therefore, α-1,2-linked substitutions can interact more effectively with the glycosidic bonds and restrict the range of allowed values for the torsion angles φ and ψ. In other words, substitutions at positions α-1,2 sterically reduce the conformational fluctuations of the xylan backbone, which in turn promote stronger xylancellulose binding. Steric interactions between substitutions and the main chain of polysaccharides have previously been shown to restrict the range of allowed glycosidic angles φ and ψ.29 The conspicuous hydrogen bonding between O5 and HO3 across the glycosidic linkage in xylan may also contribute to stabilize the two-fold screw axis. Substitution at O3, such as α1,3-Ara and α-1,3-GlcA, in which the O5-HO3 bond is lost, renders the backbone less rigid compared to GAX, but as flexible as UX, in which these bonds are present. A recent study shows that xylan and substituted oligosaccharides present complex conformational free energy landscapes in solution and that the effect of hydrogen bonding for stabilization of the glycosidic linkage conformation is limited in water.30

Binding of multiple GAX chains and role of Ca2+ ions Two GAX chains cooperate to interact with cellulose. In our previous work, we have shown that two conifer xylan chains (GAX) can simultaneously bind to the cellulose surface side-byside in a twofold screw conformation, as shown in Fig. 1B-C, and that xylan-xylan cooperative effects stabilize the binding.14 Using the same set of simulations, we examined in detail the behavior of the two GAX chains on the cellulose surface. As shown in Fig. 6A-C, two GAX 12 ACS Paragon Plus Environment

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chains exhibit lower mobility, higher binding strength (as measured by the number of xylosyl units in contact with cellulose) and more stretched out conformations (as measured by the reduced number of contacted glucan chains) than single isolated chains (see Fig. 2A-C). The simulations also indicate that the 4-OH group of GlcA interacts mostly with the solvent (Fig. S1). To investigate this stabilization effect further, we simulated the binding of two UX chains placed in parallel. The results (Fig. 6D-F) show that, although two UX are less stable than two GAX on the surface of cellulose, they are more stable than a single UX (see Fig. 2D-F), indicating that interactions between the xylan backbones themselves influence their binding to cellulose. Such interactions consist of stacking of the xylosyl rings, similarly to two glucan chains in adjacent layers in the Iβ crystalline cellulose.18

Ca2+ ions cross link two xylan chains on cellulose. Since two GAX chains bind more strongly to cellulose than two UX chains, we conclude that substitutions are expected to be important to the cooperative effect. The α-1,2-GlcA substitutions carry a negative charge, so they, in principle, would repel each other and be detrimental to GAX-GAX interactions. However, we observed that Ca2+ ions cross-link the carboxylate groups of two GlcA (Fig. 7A). As each GlcA bears a charge of –1, the GlcA–Ca2+–GlcA complex is electrically neutral and thus the GlcA substitutions do not effectively repel each other. Therefore, with the participation of Ca2+ ions, known to be abundant in plant cell walls31,32 and to bind to xylan,33 the cooperative GAX-GAX interactions are likely to be enhanced via Ca2+-meditated interactions between the GlcA decorations. The electrostatic interactions of the Ca2+ ions with the GlcA substitutions seem to be very stable Fig. 7B shows the distance between the GlcA substitutions and each of the two Ca2+ 13 ACS Paragon Plus Environment

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present in the system. Initially, the Ca2+ ions were placed in solution and far from the polysaccharides. Their distances to the GlcA substitutions are high (up to ~60 Å) in the early stages of the simulations. Once the Ca2+ ions encounter the GlcA substitutions and GlcA–Ca2+– GlcA complexes are formed, the GlcA–Ca2+ distances remain strictly around 2.2 Å and the Ca2+ ions no longer migrate back to the bulk during the remainder of the simulation time. Hence, the GlcA-mediated cooperative effect is strongly influenced by the presence of Ca2+. The simulations also indicate that the Ara substitutions interact with the main chain of the adjacent xylan via, hydrogen bonds. However, such interactions are highly dynamic, that is these hydrogen bonds continuously break and reform. The average fraction of hydrogen bonded Ara residue-xylan backbone pairs is only ~20% of the entire simulation time, and therefore seem to be much less important than the GlcA–Ca2+–GlcA complexes to the GAX-GAX cooperative effect. Thus, two GAX packed side-by-side cooperate to the adsorption by stacking interactions between the main chains and by the GlcA–Ca2+–GlcA complex. Both of these interactions contribute to decrease the level of xylan backbone conformational fluctuations relative to that of a single chain and, therefore, enhance GAX adsorption on the cellulose surface.

With three xylan chains, pairs of GlcA are bridged by Ca2+ ions. These results raise the question of whether a cellulose microfibril could be entirely coated by GAX. As shown in Fig 7A, Ca2+ ions can cross link two GlcA substitutions. According to this picture, if there was a third xylan chain in parallel, one of the GlcA would not participate of the calcium bridge, so that only an even number of xylans would likely be able to stably bind to the cellulose surface. To investigate this possibility, we simulated a system containing three GAX chains laid in parallel on the hydrophilic surface of cellulose and sufficient Ca2+ ions to render the system electrically neutral. The results displayed in Fig. 8A-C show that the GAX chains are highly stable on the 14 ACS Paragon Plus Environment

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cellulose surface with low rmsd values, high percentage of bound xylosyl units, and number of contacted glucan chains representative of stretched out conformations. Figure 8D shows the mobility of the middle xylan chain considering only the central xylosyl residues. The mobility of this xylan chain is similar to that of a glucan chain adsorbed on the cellulose (Fig. 2G), so that, in an entirely coated cellulose microfibril, the GAX chains would appear with the same mobility of cellulose glucan chains. The Ca2+ ions cross link two of the three GlcA and coordinate the third GlcA (Fig. 8E). Thus, a fourth GAX chain could bind alongside the third chain and share the Ca2+ ion. Therefore, it is possible that GAX coats the whole cellulose microfibril in the presence of Ca2+ ions between pairs of aligned GlcA substitutions. Depletion of Ca2+ ions would weaken GAX adsorption on cellulose.

Xylan-cellulose interactions at high temperatures In this section, we investigate the behavior of GAX on cellulose at a temperature corresponding to what is commonly employed in hydrothermal pretreatment of plant biomass (160 °C). Effects of the environment ionic strength are also investigated because of its importance in industrial settings.

GAX chains move to the hydrophobic face of cellulose. The general behavior of a single GAX chain at 160 °C is shown in Fig. 9A-C. No full dissociation of the GAX chain from the cellulose surface was observed in any the triplicate runs, as evident from the high percentage of bound xylosyl residues as a function of the time (Fig. 9B). Instead, GAX becomes quite mobile in the beginning of the simulation and slides from the hydrophilic to the hydrophobic surface, assuming a well-defined binding mode that resembles the crystalline packing of the glucan chain in the cellulose structure (Fig. 9D). This can be seen by the rmsd analysis (Fig. 9A), which 15 ACS Paragon Plus Environment

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shows high mobility in the beginning of the simulation and a pronounced stabilization after this transient period, which varies according to the simulation run. Moreover, the stable interaction of GAX with the cellulose hydrophobic surface results in no fluctuation in the number of glucan chains contacted by the xylan after it has reached this surface (Fig. 9C), which reflects the stretched conformation of the chain. Once GAX gets to the hydrophobic face, it remains there during the remaining simulation time. This was further corroborated by extending one of these simulations from 200 to 500 ns (see Fig. S5). Hence, at high temperatures, GAX has higher affinity for the hydrophobic face than for the hydrophilic face. When two GAX are adsorbed in parallel, we observe no dissociation of the GlcA–Ca2+– GlcA complex (see Fig. S6) at both ambient and high temperatures, so that the two GAX remain bridged to each other by the Ca2+ ions. Up to 200 - 300 ns of simulation time, the pair of GAX chains is very mobile but does not reach the hydrophobic face, indicating that the system exhibits a slower dynamics than the single GAX system. After ~300 ns one of the GAX chains (Fig. 10, in blue) moves to the cellulose hydrophobic face and binds in a stretched out conformation, similar to a single GAX. The second GAX (Fig. 10, in red) remains bound to the other GAX through the Ca2+ bridge and interacts more loosely with the hydrophobic face, as indicated in Fig. 10D, which shows representative snapshots of the GAX chains during the course of the simulations.

Salt clusters are formed around the GlcA substitutions at high ionic strengths. Finally, in order to assess the effect of a higher ionic strength, we performed additional simulations (at 300 K and 433 K) of a system comprised by two GAX chains bound to cellulose and immersed in a CaCl2 solution containing 8 Ca2+ and 12 Cl–, instead of a solution containing only two Ca2+ ions to neutralize the negative charges of the GlcA substitutions. The simulations at 433 K (160 oC) 16 ACS Paragon Plus Environment

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showed that GlcA can nucleate the formation of salt clusters in its vicinity (Fig. 11), possibly further strengthening the GAX-GAX interactions. At room temperature, such salt clusters are not observed within 200 ns of MD (data not shown) and only the Ca2+ interact with the GlcA substitutions, as portrayed in Fig. 7A. The reason for this effect is that at such high temperatures, the water dielectric constant decreases considerably and water loses its strongly polar solvation capacity,34,35 so that the ions tend to aggregate around the GlcA substitutions. Therefore, our simulations suggest that high ionic strength can contribute to keep GAX chains bound to each other under pretreatment conditions, thereby increasing the biomass recalcitrance.

Discussion Xylans containing Ara and GlcA substitutions (GAX) are naturally found in conifer, grass and dicot cell walls.12 In all cases, Ara substitutions are linked to the xylan backbone by α1,2 and/or α-1,3 bonds, whereas the GlcA substitutions are linked by α-1,2 bonds only.13,36,37 Using MD simulations, we show that such substitutions influence how GAX interacts with cellulose. The position of link between the substitutions and the xylan main chain (α-1,2 or α-1,3 bonds), rather than the chemical nature of substitution, determines how the GAX chain interacts with the cellulose hydrophilic surface. α-1,2-linked substitutions seem to stabilize cellulosexylan interactions by restraining the twofold screw xylan backbone conformation while lying on the cellulose surface. Other effects may play roles in determining the conformational free energy landscape of xylan adsorbed on cellulose and further investigations are necessary. Nevertheless, these conclusions are supported by previous findings that xylans lacking α-1,2-GlcA substitutions are more easily extracted from Arabidopsis cell walls than xylans containing α-1,2GlcA.38 In addition, α-1,2-GlcA and acetyl substitutions (linked at positions 2 and 3) have been


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shown to have some functional equivalence in plant cell walls in vivo,39 further corroborating that the type of link is more important than chemical nature of the substitution. α-1,3 substitutions do not significantly affect xylan-cellulose interactions, as revealed by the comparison between unsubstituted (UX) and α-1,3-substituted xylans adsorbed on cellulose. Thus, α-1,3-Ara substitutions naturally found in plants may be related to other functions in the cell wall, such as xylan cross-linking through feruloylation (which occurs via α-1,3-Ara in grasses), association with lignin to form lignin-carbohydrate complexes, and modulation of xylan solubility in the cell wall.37 The present results contrast to our previous simulations of xylans bound to the (010) and (020) hydrophilic faces of a square cellulose fibril, in which no significant effects of acetyl and GlcA substitutions were observed on xylan-cellulose interactions when compared to UX.15 Possibly, the stacking interactions established between the xylan and glucan chains in the (010)/(020) faces we used in our previous simulations – which, unlike the (110) face, exhibit well-defined grooves – are strong enough to mask the effects of the substitutions that are found here. When multiple GAX chains are bound to the cellulose surface, enhanced binding stability due to GAX-GAX interactions was observed, which are comprised of stacking interactions between the xylan backbone residues and Ca2+-mediated cross linking between GlcA substitutions. It is well known that Ca2+ ions are essential for the cell wall structural properties, but the role of Ca2+ ions has been attributed to their association with pectin, and not with xylans.31,32 According to our results, it is possible that trace amounts of Ca2+ ions also play roles in the stabilization of xylans on the cellulose surface, allowing the complete coating of cellulose by negatively charged GAX chains. The complete coating of cellulose by hemicellulose has been previously suggested to happen in cell walls,16,19 with important implications for the arrangement


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of the cellulose microfibrils in native plant cell walls, for instance, by preventing cellulose coalescence and facilitating or reducing the formation of macrofibrils. We have also simulated GAX-cellulose complexes at high temperature to obtain a picture of the molecular events induced by biomass hydrothermal pretreatments. Single or multiple GAX initially on the hydrophilic face move to the hydrophobic face under hydrothermal pretreatment conditions. In such process, the Ca2+ ions keep the GAX chains cross-linked on the hydrophobic face, and this is further enhanced under elevated ionic strength of the solvent environment, with the formation of salt clusters around the GlcA substitutions. Our model represents the tightly bound oligomeric xylan fraction that remains in biomass after the pretreatments,40,41 and the simulations suggest that Ca2+ ions can play roles in xylan-cellulose interactions during pretreatments. Such tightly bound GAX, even in small amounts, have inhibitory effects on the enzymatic hydrolysis of biomass by creating physical barriers to the enzymes.42 This is especially true for cellobiohydrolases, which act directly on the hydrophobic face of crystalline cellulose.43 The fact that xylans are preferentially located in the hydrophobic surface of cellulose, with their backbones aligned in parallel and highly ordered, suggests that the hydrophilic faces of cellulose would be more available to interact with each other and aggregate upon heating, as indicate our recent predictions.44 (Although we have not performed additional simulations lowering the temperature back to room temperatures after the simulated pretreatment conditions, we expect the xylan would remain on the hydrophobic surface even if the temperature were lowered again because of the high energy barrier involved in rehydrating as the hydrophobic contacts between xylan and cellulose are broken.44,45) Therefore, hydrothermal pretreatments seem to eliminate xylans from the hydrophilic face and induce cellulose-cellulose aggregation via this face, resulting in a structure consisting of bundles of cellulose microfibrils


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with the hydrophobic surface coated with xylan chains. FT-IR and solid-state NMR experiments support these conclusions.46,47 Xylan adsorption on cellulose has been evaluated in vitro, but the conclusions should not be directly compared with our study. For instance, in contrast to our results, it has been shown that low degree of xylan substitution relates to a more efficient xylan adsorption on the cellulose surfaces.48,49 In such adsorption experiments, the equilibrium between xylan in the adsorbed and liquid phases is measured, and not the direct interaction between the cellulose and xylan chains. Increasing the solution concentration of xylans can, for instance, lead to self-assembly and formation of nanoparticles that will entirely adsorb on cellulose.50,51 If the degree of substitution is low, these aggregates are stable and accumulate on the cellulose surface. Here, we study the specific interaction of xylan chains with cellulose and do not take in account the effect of xylan concentration in the surrounding medium.

Conclusions We performed a large set of molecular dynamics simulations to investigate how xylans decorated with Ara and GlcA branches interact with the (110) hydrophilic face of cellulose. The simulations showed that the substitutions stabilize the xylan binding to cellulose. For single xylan chains on cellulose, α-1,2-substitutions can stabilize xylan-cellulose interactions by restricting the mobility of the xylan backbone on cellulose – an effect that is not dependent on the type of substitution. In the case of multiple xylans on cellulose, Ca2+-mediated cross-linking between GlcA substitutions strongly stabilize stretched out conformations of xylan chains. At high temperature, xylans do not dissociate from cellulose, but are preferentially adsorbed onto the (100) hydrophobic face instead, where cluster of ions surrounding GlcA substitutions can


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strongly enhance interchain interactions. These results explain unexpected roles of Ara and GlcA xylan substitutions and provide insights to enhance the current understanding of the plant cell wall architecture and its modification by thermochemical pretreatments.

Associated Content Supporting Information. Additional graphs and simulation data on the interaction between xylan and cellulose. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information Corresponding author *E-mail: [email protected] Notes: The authors declare no competing financial interest.

Acknowledgements We thank the Sao Paulo Research Foundation for supporting this work (Grants # 2013/08293-7, 2014/10448-1 and 2015/25031-1). All calculations were performed at the Center for Computational Engineering and Sciences at Unicamp. We thank Marta Busse-Wicher for helpful discussions.


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Figures

Figure 1. Molecular models used in the simulations. (A) Glucuronoarabinoxylan model following the conifer substitution pattern.14 (B) Cross-sectional views of one and two GAX on the cellulose surface. The two GAX chains were considered in register only. Other two-chain configurations are possible. Only half of a 36-chain cellulose microfibril (in dark gray) was considered in the simulations. (C) Side views of the complexes shown in (B).


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Figure 2. Analysis of xylan on the cellulose surface. (A) Rmsd of GAX from its initial position on the cellulose surface, showing that, after a transient accommodation during the first 50 ns, the chain remains stably bound to cellulose, exhibiting only local thermal fluctuations. (B) Percentage of xylosyl residues that are bound, i.e., within a 2.5 Å distance from cellulose, during the course of the simulations. Such quantity is related to the binding strength. (C) Number of glucan chains that contact the bound xylan chain, indicating how stretched the xylan is on the cellulose surface. (D), (E) and (F): same as (A), (B) and (C), respectively, but for UX. (G), (H) and (I): same as (A), (B) and (C), respectively, but for a bound glucan chain. The different colors represent independent simulations.


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Figure 3. Analysis of xylan on the cellulose surface. Rmsd, percentage of bound xylosyl units, and number contacted glucan chains for AX with α-1,3-Ara substitutions (A, B and C), and GX with α-1,2-GlcA substitutions (D, E and F) on the cellulose surface. The different colors represent independent simulations.


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Figure 4. Analysis of xylan on the cellulose surface. Rmsd, percentage of bound xylosyl residues, and number of contacted glucan chains for AX with α-1,2-Ara substitutions (A, B and C) and for GX with α-1,3-GlcA substitutions (D, E and F).


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Figure 5. (A) Snapshot of GAX showing that the glycosidic bond near α-1,2-GlcA points towards the solution and the glycosidic bond closest to α-1,3-Ara points towards the cellulose. (B) Normalized distribution of the van der Waals energy between the glycosidic oxygen and the α-1,2-GlcA and α-1,3-Ara substitutions. Interactions of α-1,2-GlcA with the glycosidic oxygen is mostly repulsive due to very close contact.


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Figure 6. Analysis of a pair of xylan chains on the cellulose surface. Rmsd, percentage of bound xylosyl, and number of contacted glucan chains for two GAX (A, B and C) and two UX (D, E and F) on the cellulose surface. The different colors represent independent simulations.


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Figure 7. Interactions of Ca2+ ions with GAX. (A) Snapshot of two GAX chains bound to the cellulose surface, showing Ca2+ ions cross linking neighbor GlcA substitutions. (B) Distance between each of two Ca2+ ions present in the system [Ca2+ (1) and Ca2+ (2)] and the GlcA substitutions. Each panel represents a different simulation. Initially, as the Ca2+ ions are dispersed in the medium, the time history of the distance exhibits considerable fluctuations. After complexation of the Ca2+ ions by the GlcA, the distances fluctuate strictly around 2.2 Å.


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Figure 8. Analysis of three GAX on the cellulose surface. (A) Rmsd, (B) percentage of bound xylosyl and (C) number of contacted glucan chains for three GAX. (D) Rmsd of the GAX chain considering only the central xylosyl residues between the two GlcA substitutions. (E) Snapshot of three GAX bound to the cellulose surface, showing a Ca2+ ion cross linking pairs of neighboring GlcA substitutions. The different colors represent independent simulations.


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Figure 9. Analysis of a single GAX on the cellulose surface at high temperature (160oC). (A) Rmsd (relative to the structure where xylan is ideally stretched on the hydrophobic surface), (B) percentage of bound xylosyl, and (C) number of contacted glucan chains. Different colors represent different simulations. (D) Snapshots illustrating the GAX moving from the hydrophilic to the hydrophobic cellulose surface.


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Figure 10. Analysis of two GAX on the cellulose surface at high temperature. (A) Rmsd (relative to the structure where xylan is ideally stretched on the hydrophobic surface), (B) percentage of bound xylosyl, and (C) number of contacted glucan chains. Different colors represent different GAX and different panels represent different simulations. (D) Snapshots of the two GAX chains (red and blue) illustrating their displacement from the hydrophilic to the hydrophobic cellulose surface.


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Figure 11. Snapshot showing a cluster of Ca2+ and Cl– ions around GlcA substitutions at high temperature.


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Effects of Xylan Side-Chain Substitutions on XylanâCellulose

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