Supplementary Figure 5: VHHs 5D, 7F, and E3 recognize distinct epitopes and exploit different mechanisms to neutralize TcdB.
From: Structure of the full-length Clostridium difficile toxin B
(a, b) pH-dependent conformational change of TcdB1072–1433 probed by ANS. TcdB1072–1433, the TcdB1072–1433–5D complex, or 5D alone was incubated with ANS in buffers with different pH, and the peak fluorescence intensities of ANS at 474 nm are shown. Error bar represents SD of three replicate experiments. (c) The structure of the TcdB1072–1433–5D complex (light blue, residues 1090–1431 of TcdB were fully resolved in this crystal structure) was superimposed to the full length TcdB–5D complex (TcdB and 5D were colored yellow and pink, respectively). The overall Cα atom r.m.s.d. is ~0.608 Å. (d) Structural superposition to compare the conformations of the pore-forming region among the TcdB1072–1433–5D complex that was crystallized at pH 8.5 (light blue), the TcdB–5D complex that was crystallized at acidic pH (yellow), and TcdA at neutral pH (orange). (e, f) A close-up view into the interface between TcdB and 5D in the structure of the TcdB1072–1433–5D complex, where the CDR2 of 5D (e), as well as its CDR1 and CDR3 (f) bind TcdB with extensive interactions. (g) 7F fixes the conformation of the Helix 1 (residues 525–539) and the Helix 2 (residues 137–158) in the GTD, which are in close proximity to the active site of the CPD and the scissile bond (L544–G545). GTD, CPD, and 7F are colored red, light blue, and green, respectively. The peptide linker between the GTD and the CPD is colored blue. A close-up view of the boxed area is shown in (h). (i) Detailed interactions between GTDVPI10463 and 7F. The interacting residues are colored green and pink for 7F and GTD, respectively. The 7F-binding interfaces are largely identical between GTDVPI10463 and GTD. (j) Interactions between GTD and E3. The residues involved in interactions are colored wheat and pink for E3 and GTD, respectively.
