Carbohydrate binding module recognition of xyloglucan defined by polar contacts with branching xyloses and CH‐Π interactions

Abstract

Engineering of novel carbohydrate-binding proteins that can be utilized in various biochemical and biotechnical applications would benefit from a deeper understanding of the biochemical interactions that determine protein-carbohydrate specificity. In an effort to understand further the basis for specificity we present the crystal structure of the multi-specific carbohydrate-binding module (CBM) X-2 L110F bound to a branched oligomer of xyloglucan (XXXG). X-2 L110F is an engineered CBM that can recognize xyloglucan, xylans and β-glucans. The structural observations of the present study compared with previously reported structures of X-2 L110F in complex with linear oligomers, show that the π-surface of a phenylalanine, F110, allows for interactions with hydrogen atoms on both linear (xylopentaose and cellopentaose) and branched ligands (XXXG). Furthermore, X-2 L110F is shown to have a relatively flexible binding cleft, as illustrated in binding to XXXG. This branched ligand requires a set of reorientations of protein side chains Q72, N31, and R142, although these residues have previously been determined as important for binding to xylose oligomers by mediating polar contacts. The loss of these polar contacts is compensated for in binding to XXXG by polar interactions mediated by other protein residues, T74, R115, and Y149, which interact mainly with the branching xyloses of the xyloglucan oligomer. Taken together, the present study illustrates in structural detail how CH-π interactions can influence binding specificity and that flexibility is a key feature for the multi-specificity displayed by X-2 L110F, allowing for the accommodation of branched ligands.

Advances in molecular engineering of carbohydrate-binding modules

“…These results suggest the relevance for these interactions on the substrate specificity of CBMs. 128 Recently, the neutron structure of the same mutant, specifically X-2 L110F derived from CBM4-2 from the R. marinus xylanase, was reported, 129 providing the first CBM structure define using this technique and making it possible to demonstrate the importance of water molecules in hydrogen bonding in CBM-ligand interactions, a previously questioned issue. It is known that both the parental CBM X-2 and its X-2 L110F mutant contain ordered water molecules at the binding site and that at least 10 of these molecules are displaced by the union with a carbohydrate, although three of these molecules remain at the binding site.…”

Crystallization, neutron data collection, initial structure refinement and analysis of a xyloglucan heptamer bound to an engineered carbohydrate-binding module from xylanase

“…At 1.0 Å resolution we were able to observe unassigned F o À F c density corresponding to some H atoms in the protein, in the ligand and on a few well ordered water molecules. However, the data were not strong enough to support the modelling of explicit positions for H atoms (von Schantz, Hå kansson et al, 2014;von Schantz et al, 2012). From these previous ultra high-resolution X-ray crystallographic studies, many details of carbohydrate binding were observed.…”

“…X-2 is specific for xylan (and its synthetic mimic oligoxylose). Further mutagenesis identified the single mutant X-2 L110F (von Schantz et al, 2012) that, similar to the wild-type module, is able to bind multiple carbohydrates including not only xylan but also -glucan and xyloglucan (von Schantz, Hå kansson et al, 2014;von Schantz et al, 2012). In both CBM variants residue 110 stacks against the ligand in the cavity of the CBM ligand-binding site (Fig.…”

“…1). Importantly, X-2 L110F (and its immediate predecessor X-2), in contrast to other similar modules (including CBM4-2), crystallizes sufficiently well to allow the development of CBM-ligand complex crystals (Gullfot et al, 2010;von Schantz et al, 2012;von Schantz, Hå kansson et al, 2014).…”

A Mini-review on the Applications of Cellulose-Binding Domains in Lignocellulosic Material Utilizations

“…This wedgeshaped structure is likely to be shared by all fungal CBDs (Mattinen et al 1997). The specificity of CBMs can be changed through simple mutations (von Schantz et al 2014). It was suggested that tryptophan residues contribute to higher binding affinity than tyrosine residues.…”