Figure 4.

Alternative Y11 χ₁ angle rotamer supported by epitope mapping. (A) R-factor ratios comparing epitope maps based on theoretical STD enhancements and the experimental STCD_AF-CRL epitope map. Theoretical epitope maps were generated based on the NTS₁ crystal structure (PDB 4BUO); based on the averaged STD enhancements over four MD trajectories (MD_av) of NT10-13 and NT8-13 bound to enNTS₁model; and based on the individual cluster representatives (clusters 1–10) extracted from the NT10-13 and NT8-13 bound MD trajectories. (B) Epitope map based on STD enhancements of NT10-13 MD cluster representative 1 normalized to the maximum value. This cluster showed the lowest R-factor (R = 0.40) among the theoretical epitope maps generated. The corresponding values are P10 H^α, 22%; P10 H^β2, 23%; P10 H^γ2, 17%; P10 H^β2/γ3, 48%; Y11 H^β2, 66%; Y11 H^β3, 65%; Y11 H^δ1/δ2, 56%; Y11 H^ε1/ε2, 40%; I12 H^β, 26%; I12 H^α/L13 H^α, 21%; L13 H^β2/β3/H^γ, 100%; and L13 H^δ1, 48%. (C) Close-up of the ECL2 region of NT10-13 MD cluster representative 1 showing that the Y11 side chain adopts a χ₁ g^– conformation. (D) Close-up of the ECL2 region of 4BUO showing the Y11 side chain in a χ₁ g⁺ conformation. Close-ups have been reduced to residues interacting with Y11 for clarity (two-dimensional ligand maps showing all receptor residues interacting with NT10-13 given in Figure S12). (E and F) Rotamer χ₁ angle of Y11 in four independent MD simulations of NT10-13 and NT8-13, respectively, bound to enNTS₁model.