Fig. 3: Computational design and experimental verification results from affinity maturation design of Nb01.
From: Targeting Ras-binding domain of ELMO1 by computational nanobody design
a Comparison of ΔFlexddG and MM/PBSA between Nb01 sequence variants selected (red) and unselected (grey) by binding affinity estimation with reference to Nb01 (green) and Nb02 (black). b Relative positions in local sequence space among Nb01 sequence variants selected (red) and unselected (grey) as the final candidates from affinity maturation design with reference to Nb01 (green) and Nb02 (black). c Spatial overlap between the final candidates from affinity maturation design with RhoG according to the modeled nanobody-ELMO1-RBD complexes. d Comparison of SPR sensorgrams of Nb29 and Nb01 at 2 μM concentration (n = 2 independent experiments). e Measured KD values between the final candidates from affinity maturation design to ELMO1-RBD from SPR binding assay. Asterisks represents KD > 1e–5 M. f The newly designed mutations after affinity maturation (pink) in Nb29 were distant from the interface with ELMO1-RBD. g Comparison of loop flexibility of CDR2 (top) and CDR3 (bottom) between Nb29apo (pink) and Nb01apo (cyan) from molecular dynamics simulation. Locations of Cα atoms of the interface residues (black) and CDR-framework designed residues (color of the respective design) were annotated on the CDR loops. For the line plots, the solid lines and the shaded regions represent the mean and the range of RMSD of the CDR loops in snapshots extracted from independent production runs (n = 5), respectively. h Binding of Nb29 to ELMO1 suppressed RhoG/ELMO1-RBD interaction. Competitive binding assays comparing SPR sensorgrams of ELMO1-RBD without and with an excess of Nb29 against immobilized active RhoG. The concentrations of ELMO1-RBD and Nb29 were 5 μM and 500 μM, respectively. i Normalized binding of ELMO1-RBD to active RhoG. Data are means ± s.d. (n = 3 independent experiments).
