Volume 11, Issue 6, Pages 665-675 (June 2003) Structural and Thermodynamic Dissection of Specific Mannan Recognition by a Carbohydrate Binding Module, TmCBM27  Alisdair B. Boraston, Timothy J. Revett, Catherine M. Boraston, Didier Nurizzo, Gideon J. Davies  Structure  Volume 11, Issue 6, Pages 665-675 (June 2003) DOI: 10.1016/S0969-2126(03)00100-X

Figure 1 Isotherms of TmCBM27 Binding to Mannotetraose (A) and Mannohexaose (B and C) Obtained by Isothermal Titration Calorimetry (A and B) The titration of mannooligosaccharides into TmCBM27 (150 μM). The solid line through the data in (A) shows the fit of the data to a bimolecular interaction model, whereas the solid line in (B) shows the trend only. (C) The titration of TmCBM27 (1.6 mM) into mannohexaose (150 μM). The solid line through the data shows the fit to binding model treating mannohexaose as a divalent acceptor. Structure 2003 11, 665-675DOI: (10.1016/S0969-2126(03)00100-X)

Figure 2 Association of TmCBM27 with Mannohexaose as Measured by Dynamic Light Scattering (A) The change in measured molecular weight with the addition of mannohexaose. The solid line represents the fit of the data by nonlinear regression to the proposed binding model (see text). Error bars represent the standard error of duplicate sample. (B) The dependency of monomer (filled symbols) and dimer (open symbols) species concentrations on mannohexaose addition for the experimental data (circles) compared with the theoretical values calculated from the model (squares). Structure 2003 11, 665-675DOI: (10.1016/S0969-2126(03)00100-X)

Figure 3 Proposed Equilibria Describing Mannohexaose Recognition by TmCBM27 P, TmCBM27; L, mannohexaose. Ka,m and Ka,d are the equilibrium association constants for the formation of the monomeric complex and the dimeric complex, respectively. Structure 2003 11, 665-675DOI: (10.1016/S0969-2126(03)00100-X)

Figure 4 Isotherm Obtained by the Titration of TmCBM27 (2.53 mM) into Carob Galactomannan (0.19 g/l) as Measured by ITC Data is present in the standard format of energy evolved per injection (A) and in Scatchard form (B). The solid line in (A) shows the trend only. Structure 2003 11, 665-675DOI: (10.1016/S0969-2126(03)00100-X)

Figure 5 Three-Dimensional Structure of Uncomplexed TmCBM27 (A) The overall secondary structure of the protein is shown with the aromatic amino acids in the binding site shown in “licorice.” The bound metal ion is shown as a blue sphere. Solvent-accessible surfaces of TmCBM27 complexed with (B) G2M5 and (C) mannohexaose. Purple regions indicate the surface contributed by the binding site aromatic residues. The sugar molecules are shown in blue and red licorice. Structure 2003 11, 665-675DOI: (10.1016/S0969-2126(03)00100-X)

Figure 6 Observed Electron Density for G2M5 (A) and Mannohexaose (B) Bound to TmCBM27 All maps are maximum-likelihood (Murshudov et al., 1997)/σA-weighted (Read, 1986) 2Fobs – Fcalc electron density maps contoured at 0.55 (G2M5) and 0.43 (mannohexaose) electrons/Å3. Aromatic residues involved in binding are shown in licorice. Structure 2003 11, 665-675DOI: (10.1016/S0969-2126(03)00100-X)

Figure 7 A Schematic Showing the Interactions of TmCBM27 with G2M5 (A) and Mannohexaose (B) Hydrogen bonds, dashed lines; water molecules, filled circles. Binding subsites referred to in the text are shown underneath the schematics with brackets. Structure 2003 11, 665-675DOI: (10.1016/S0969-2126(03)00100-X)