Structural basis for carbohydrate-binding specificity—A comparative assessment of two engineered carbohydrate-binding modules

Abstract

Detection, immobilization and purification of carbohydrates can be done using molecular probes that specifically bind to targeted carbohydrate epitopes. Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that can be engineered to bind and detect specifically a number of carbohydrates. Design and engineering of CBMs have benefited greatly from structural studies that have helped us to decipher the basis for specificity in carbohydrate-protein interactions. However, more studies are needed to predict which modifications in a CBM would generate probes with predetermined binding properties. In this report, we present the crystal structures of two highly related engineered CBMs with different binding specificity profiles: X-2, which is specific for xylans and the L110F mutant of X-2, which binds xyloglucans and β-glucans in addition to xylans. The structures of the modules were solved both in the apo form and complexed with oligomers of xylose, as well as with an oligomer of glucose in the case of X-2 L110F. The mutation, leucine to phenylalanine, converting the specific module into a cross-reactive one, introduces a crucial hydrogen-π interaction that allows the mutant to retain glucan-based ligands. The cross-reactivity of X-2 L110F is furthermore made possible by the plasticity of the protein, in particular, of residue R142, which permits accommodation of an extra hydroxymethyl group present in cellopentaose and not xylopentaose. Altogether, this study shows, in structural detail, altered protein-carbohydrate interactions that have high impact on the binding properties of a carbohydrate probe but are introduced through simple mutagenesis.

Bioinspired assemblies of plant cell wall polymers unravel the affinity properties of carbohydrate-binding modules

“…His-tagged CBMs were produced in T7 Express E. coli cells (New England Labs, Ipswich, England), as previously reported. 39 Purification of the proteins from cell suspensions after sonication was performed using the Ni-NTA matrix (Qiagen, Hilden, Germany) according to previously reported protocols. 39 Preparation and characterization of fluorescent CBMs (probes) Covalent grafting of fluorescein isothiocyanate (FITC) onto different CBMs can only occur with amine functions of the N-terminal residue and/or Lys residues.…”

“…39 Purification of the proteins from cell suspensions after sonication was performed using the Ni-NTA matrix (Qiagen, Hilden, Germany) according to previously reported protocols. 39 Preparation and characterization of fluorescent CBMs (probes) Covalent grafting of fluorescein isothiocyanate (FITC) onto different CBMs can only occur with amine functions of the N-terminal residue and/or Lys residues. Analysis of the amino acid sequence 32 of each CBM shows that all modules have a single Lys residue at position 55 providing a total of two and three such primary amino groups per monomeric and dimeric CBM, respectively.…”

“…3D structural visualization of the CBMs was performed with the previously solved structures of CBM X-2 (complexed with xylopentaose; PDB ID: 2Y6L) 39 and CBM4-2 (PDB ID: 1K45) 40 using PyMOL Molecular Graphics System, Version 1.3 (Schrödinger LLC, Portland, OR, USA).…”

“…CBMs were run on 12.5% non-denaturing polyacrylamide gels with or without 0.5% FAXs as previously reported. 39 Isothermal titration calorimetry (ITC)…”

“…The protocol has been described previously. 39 Briefly, a MicroCal VP-ITC system (Microcal (LLC), Northampton, MA, USA) was used to monitor heat exchange at 302 K and 300 rpm of 30 injections of 10 mL of the ligand (first injection 5 mL) with an interval of 300 seconds. CBM solutions in 20 mM HEPES/100 mM NaCl/5 mM CaCl 2 pH 7.5 buffer had an initial protein concentration of 50 mM.…”