Regular Motifs in Xylan Modulate Molecular Flexibility and Interactions with Cellulose Surfaces

Abstract

Xylan is tightly associated with cellulose and lignin in secondary plant cell walls, contributing to its rigidity and structural integrity in vascular plants. However, the molecular features and the nanoscale forces that control the interactions among cellulose microfibrils, hemicelluloses, and lignin are still not well understood. Here, we combine comprehensive mass spectrometric glycan sequencing and molecular dynamics simulations to elucidate the substitution pattern in softwood xylans and to investigate the effect of distinct intramolecular motifs on xylan conformation and on the interaction with cellulose surfaces in Norway spruce (Picea abies). We confirm the presence of motifs with evenly spaced glycosyl decorations on the xylan backbone, together with minor motifs with consecutive glucuronation. These domains are differently enriched in xylan fractions extracted by alkali and subcritical water, which indicates their preferential positioning in the secondary plant cell wall ultrastructure. The flexibility of the 3-fold screw conformation of xylan in solution is enhanced by the presence of arabinofuranosyl decorations. Additionally, molecular dynamic simulations suggest that the glycosyl substitutions in xylan are not only sterically tolerated by the cellulose surfaces but that they increase the affinity for cellulose and favor the stabilization of the 2-fold screw conformation. This effect is more significant for the hydrophobic surface compared with the hydrophilic ones, which demonstrates the importance of nonpolar driving forces on the structural integrity of secondary plant cell walls. These novel molecular insights contribute to an improved understanding of the supramolecular architecture of plant secondary cell walls and have fundamental implications for overcoming lignocellulose recalcitrance and for the design of advanced wood-based materials.

Figure 1.

Enzymatic profiling of spruce xylan. A, General structure of AGX. B, Substrate recognition pattern of the xylanolytic enzymes used for AGX digestion, based on previous literature (Pell et al., 2004; Vardakou et al., 2008; Pollet et al., 2010; St John et al., 2011). The position of the hydrolytic activity is marked with a wedge at the glycosidic linkage between the −1 and +1 subsites. C, Oligosaccharide fingerprinting with high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the spruce AGX-A. D, Oligomeric mass profiling with ESI-MS of the AGX-A. Note that P refers to a pentose (Xyl or Ara) and U^m refers to mGlcA.

Figure 2.

Comprehensive MS sequencing of selected oligosaccharides from spruce xylan. A, SIM chromatograms of the oligosaccharides released by the GH30 glucuronoxylanase alone (in black) or in combination of a GH51 arabinofuranosidase (in blue). B, Oligosaccharide fragmentation by MS/MS (fragment assignation according to Domon and Costello, 1988). C, Classification of the oligosaccharide motifs in spruce xylan based on the substitution pattern.

Figure 3.

A, Nomenclature of the glycosidic linkages (GL) of the simulated XOs. B, Definition of dihedral angles in the backbone GLs: φ = O5′-C1′-O4-C4 and ψ = C1′-O4-C4-C3. For the GL connecting Ara to the xylopyranose backbone, the dihedral angles are defined as φ = O4′-C1′-O3-C3 and ψ = C1′-O3-C3-C2, and for the mGlcA GLs, φ = O5′-C1′-O2-C2 and ψ = C1′-O2-C2-C1. C, A xylooligomer (gray) on a (1-10) cellulose surface (green). D, A xylooligomer (gray) on a (200) cellulose surface. The views are directed along the cellulose chain axis (from the nonreducing end to the reducing end).

Figure 4.

Conformation of XOs in water solution. A, Division of the free energy surfaces into regions [1, 2, 3⁽⁺⁾, 3⁽⁻⁾, 4⁽⁺⁾, and 4⁽⁻⁾]. B, Free energy surfaces (φ, ψ) for the backbone glycosidic linkages in X₆ and XXA³XU^mX. The probabilities (%) for each region within each free energy surface are presented, and the se for the probabilities is 0.0 to 1.6. C, Structure and conformation of X₆ and XXA³XU^mX. For the 2₁-fold conformation, the glycosidic linkages are (280,° 80°), and for the 3₁-fold conformation, they are (290°, 130°). The linkage connecting the Ara side group is (295°, 250°), and that for mGlcA is (80°, 90°), which are common minima for the respective side groups (Supplemental Fig. S15).

Figure 5.

Conformations of XOs located on cellulose surfaces. A, Free energy surfaces of X₆, XXA³XU^mX, and XXXU^mU^mX. The energy bar and notation of the glycosidic linkages are the same as in Figures 3 and 4. B, Snapshots of XXA³XU^mX and XXXU^mU^mX on (1-10) and (200) surfaces. Additional snapshots from the simulations of XOs on cellulose surfaces are presented in Supplemental Figures S17 to S19.

Figure 6.

Pulling-out experiments of the different XOs from the hydrophilic (1-10) and hydrophobic (200) cellulose microfibril surfaces. The diagrams exhibit the pulling mechanical free energies (PMF; in kJ mol⁻¹) for X₆, XXXXU^mX, XXA³XU^mX, and XXXU^mU^mX (in protonated and deprotonated forms) as a function of the distance (in nm) from the celluloses surfaces.