Fig. 5
From: Structural basis for antibody recognition of the proximal MUC16 ectodomain
The binding modes of the 4H11-scFv rearrange the CDRs of the scFv. (a, b) The overall superpositions of the structures of the unbound and MUC16-target bound 4H11-scFv. The unbound VH*-VL* (grey), bound VH (skyblue)-VL (pink), MUC16-target (orange). The view directions in 5a and b are similar to those shown in Figs. 3a and 90° rotation of the complex about a horizontal axis (5b). (c, d) The close-up views of the interfaces between VH-VH* (c) or VL-VL* with the movement indicated by the red dotted arrow. The aromatic ring of Y108 with 2Fo-Fc map was moved to the left up to ∼ 3.8 Å and The OG of S 180 with 2Fo-Fc map was also moved to the left up to ∼ 2.5 Å. (e-g) The close-up views of the hydrogen bonds formed by water molecules (W1, W2, or W3) during the complex. Y16th and G 17th (cyan) of MUC16-target (f) and S 15th, S 23rd and R 24rd (orange) of MUC16-target (g) formed the multi-hydrogen bonds with VL S180 and VH Y108/D106. See Table S4 for detailed interactions. (h) Pull-down assay was performed using 4H11-scFv and mutants as a prey and the MBP-tagged MUC16 (26 residues) as a bait. After binding, the MBP resins were washed three times, and the bound proteins were released and subjected to SDS-PAGE. Lane 1–5: input proteins of MBP- tagged MUC16-target (lane 1), 4H11-scFv (lane 2), and the scFv mutants (lane 3–5); lane 6, pull- down as a control; lane 7–8, 4H11-scFv containing VH double mutations (S53A/D106A) or VL double mutations (Y246A/N247A); lane 9, the two double mutations (S53A/D106A, Y246A/N247A) of the scFv. The residues of MUC16 (S 15th, S 22nd, R 24th, D 25th, L 26th, and Q 30th) in orange color form the hydrogen bonds or salt bridges with VH residues (S 52, S 53, A 54, N 103, D 106, and Y 108) in blue color, while the residues (Y 16th, G 17th, D 18th, and L 20th) of MUC16 in cyan color form the hydrogen bonds with VL residues (S 180, Y 246, N 247 and L 248) in pink color. (g-h) Interacting residues are labeled in close-up views of the interfaces. See Table S2, Fig S3 and S4 for versions of the detailed interactions