Journal: The Journal of Biological Chemistry
Article Title: Mechanism of Action and Epitopes of Clostridium difficile Toxin B-neutralizing Antibody Bezlotoxumab Revealed by X-ray Crystallography
doi: 10.1074/jbc.M114.560748
Figure Lengend Snippet: Crystal structure of the N-terminal half of the TcdB CROP domain bound to two bezlotoxumab Fab fragments. A, side view showing parallel binding of the two Fab fragments (Fab1 and Fab2) to their respective epitopes, E1 and E2. LRs are shown in gray and SRs in green. Fab heavy chains are shown in yellow (Fab1) and peach (Fab2) and light chains in pink (Fab1) and purple (Fab2). B, bottom-up view showing the Fab fragments bound perpendicularly to the curvature of the CROP domain. Residues of the CROP domain that directly interact with the heavy chains (yellow and peach) or light chains (pink and purple) of the Fab fragments are highlighted on the CROP surface. C, partial sequence of the light chains (Lc) and heavy chains (Hc) of bezlotoxumab, showing the six complementarity-determining regions (identified using the Molecular Operating Environment software from Chemical Computing Group); residues that interact with peptide B2 are highlighted in gray. D, sequence alignment of the two bezlotoxumab epitope regions (E1 and E2) and the two non-binding homologous regions (E3 and E4) of the TcdB CROP domain. Bezlotoxumab-interacting residues in E1 and E2 (and corresponding residues in E3 and E4) are highlighted in gray, with conserved and non-conserved substitutions (compared with E1) shown in green and red, respectively. Regions protected by bezlotoxumab in the HDX-MS experiment (Fig. 2c) are underlined, and residues putatively involved in carbohydrate binding are identified by asterisks. SRs and LRs are shown as green and gray boxes, respectively.
Article Snippet: Percentage deuteration was averaged over all time points, and significant differences between deuteration levels in the presence versus the absence of bezlotoxumab are reported in B . Mapping of the HDX-protected regions onto TcdB, as shown in , was carried out by constructing a three-dimensional model based on the crystal structure of the C-terminal fragment of TcdA ( 23 ) and using the Molecular Operating Environment computer software (Chemical Computing Group Inc., Montreal, Canada).
Techniques: Binding Assay, Sequencing, Software
