Fig. 1: Importance of the stalk and knob in the ultra-long CDR-H3.
From: Mechanistic principles of an ultra-long bovine CDR reveal strategies for antibody design
a Sequences of NC-Cow1 mutants with differences in the ultra-long CDR-H3. b Crystal structure of NC-Cow1 Fab (PDB:6OO0). c Immunoprecipitation of Expi293 supernatants after transient expression of the Fab fragments followed by SDS-PAGE. Two independent experiments. d SEC MALS chromatograms of the purified NC-Cow1 Fab variants (the molecular mass is shown in red; the value is mean of triplicates with standard deviation). e FUV CD spectra and f thermal stability of the NC-Cow1 Fab variants (the points represent the mean of triplicates with standard deviation). g Binding of 100 nM NC-Cow1 Fab variants to the immobilized HIV-1 Env protein as determined by SPR. In e, f and g, black is the wildtype (wt) NC-Cow1 Fab, orange is the mutant with deleted knob (Δknob), blue is the mutant with deleted stalk (Δstalk), and green is the mutant with a stalk made of glycine residues (G-stalk). h and i Comparative MD-simulation on the Fv of NC-Cow1 wt (h) and G-stalk (i) showing the RMSD deviation (with respect to experimental start structure) of the Fv framework alone (blue), the knob alone (orange), or the motion of the whole structure (knob plus the Fv framework, magenta). Simulation snapshots at different phases of the simulation are indicated.
