Fig 1.
Schematic models of the oligosaccharides appearing in this study.
The ligands (oligosaccharides) used for native PAGE (Fig 2) and cyclic α-nigerosyl-(1→6)-nigerose (CNN). Circles and slashed circles indicate glucose and reducing end glucose, respectively.
Fig 2.
Native-PAGE without polysaccharides (A) and in the presence of Pinedex #100 (B), dextran (C and E), and pullulan (D) are shown. The concentrations of the polysaccharides were 0.5% (w/v). For (B–E), samples containing 100 mM oligosaccharides were also used. BSA was used as a control protein. (E) The Rf values were calculated as fraction of the band position of CMMBP from BSA (offset: 0.00) to the front (1.00).
Fig 3.
ITC of CMMBP binding for CMM (A) and MM (B).
Titration thermograms (top) and binding isotherms (bottom) are shown. Assay conditions are described in the Materials and Methods.
Table 1.
Affinity and thermodynamic parameters of ligand binding to CMMBP at 25°C estimated by ITC.
Fig 4.
(A) Overall structure. The N-domain, C-domain, and hinge regions are colored in green, cyan, and magenta, respectively. Panose is shown as gray sticks. (B) Schematic presentation (top) and polder map (3.5σ) of the ligand (bottom). If the glucose unit at the nonreducing end (Glc4, indicated by a dashed-line circle) is included, the molecule becomes α-maltosyl-(1→6)-maltose (MM). (C) Stereographic view of the ligand binding site. Protein residues are colored as in panel (A). (D) Sequence conservation mapping on the molecular surface. Amino acid sequence conservation among CMMBP homologs (identity > 35%) is colored with red (high), white (middle), and blue (low).
Table 2.
Data collection and refinement statistics of the crystallography.
Fig 5.
Structural comparison of CMMBP with tmMBP3 (A), Xac-MalE (B), and Lmo0181 (C).
Cα traces of CMMBP (green for the N-domain and hinge and gray for the C-domain), tmMBP3 plus maltose (PDB ID: 6DTQ, magenta), unliganded state of tmMBP3 (6DTR, yellow), Xac-MalE (PDB ID: 3UOR, yellow) and Lmo0181 plus CNN (5F7V, magenta) are shown. The structures were superimposed with the C-domain.
Table 3.
Results of the structural similarity search using the Dali server.
Fig 6.
Comparison of the substrate binding cleft with other SBPs.
Surface representation of CMMBP (A), Lmo0181 (B, 5F7V), and TvuCMBP (C, 2ZYM) are shown. The surfaces are colored by hydrophobicity. Yellow dashed lines indicate the direction of the ligands from the nonreducing end (NRE) side to the reducing-end (RE) side. α-CD, α-cyclodextrin (cyclic glucohexasaccharide).