Solution structure, dynamics and binding studies of a family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11)

Abstract

Non-catalytic cellulosomal CBMs (carbohydrate-binding modules) are responsible for increasing the catalytic efficiency of cellulosic enzymes by selectively putting the substrate (a wide range of poly- and oligo-saccharides) and enzyme into close contact. In the present study we carried out an atomistic rationalization of the molecular determinants of ligand specificity for a family 11 CBM from thermophilic Clostridium thermocellum [CtCBM11 (C. thermocellum CBM11)], based on a NMR and molecular modelling approach. We have determined the NMR solution structure of CtCBM11 at 25°C and 50°C and derived information on the residues of the protein that are involved in ligand recognition and on the influence of the length of the saccharide chain on binding. We obtained models of the CtCBM11-cellohexaose and CtCBM11-cellotetraose complexes by docking in accordance with the NMR experimental data. Specific ligand-protein CH-π and Van der Waals interactions were found to be determinant for the stability of the complexes and for defining specificity. Using the order parameters derived from backbone dynamics analysis in the presence and absence of ligand and at 25°C and 50°C, we determined that the protein's backbone conformational entropy is slightly positive. This data in combination with the negative binding entropy calculated from ITC (isothermal titration calorimetry) studies supports a selection mechanism where a rigid protein selects a defined oligosaccharide conformation.

Enhanced features of Dictyoglomus turgidum Cellulase A engineered with carbohydrate binding module 11 from Clostridium thermocellum

“…cellulovorans CbpA (1.67 µM −1 ) 28 . The binding efficiency of chimeric Dtur CelA towards the long chains of Avicel is consistent with the results of Viegas and collaborators 19 , who observed a higher affinity of Ct CBM11 for larger ligands when compared to those with the minimal length to fit the binding cleft. This difference is probably due to the presence of more contact points in larger oligosaccharides.…”

“…These data are in accordance with those reported previously: Dtur CelA temperature optimum was stated between 70 °C and 80 °C 6 . Concerning Ct CBM11, Viegas and collaborators observed that it does not change its structure at 25 °C and 50 °C, confirming its stability in this °T range; moreover, a closer contact between Ct CBM11 and cellohexaose was detected at 50 °C than 25 °C 19 . At the most acidic pH tested, chimeric Dtur CelA showed higher relative activity compared to the native form (20% vs 0%).…”

Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy

“…Cellulose binding by type‐A CBMs is mediated by a planar array of aromatic amino acids on their surface that form stacking interactions with the sugar rings of crystalline cellulose chains resulting in strong van der Waals interactions, thus stabilizing the structure. Although many structural studies using X‐ray13–15 and solution NMR spectroscopy16–18 were conducted on CBM proteins, only a few studies employed measurements of dynamics in these systems 18–21…”

Cell wall targeted in planta iron accumulation enhances biomass conversion and seed iron concentration in Arabidopsis and rice

“…The atomistic model of the CtCBM11 (Clostridium thermocellum CBM11) was constructed using the reported NMR structure 2LR0 (Viegas et al, 2013). The blood iron-binding peptide was initially generated as a a-helix and attached to the C-terminal end of CtCBM11.…”