Fig. 4. R_AB interdomain interactions probed through MD simulations and PREs.

(A) Simulated CM distances between CBD-A and CBD-B for R_AB:Rp₂ in the A_off-B_off, A_onB_on, and hybrid A_off-B_on models with three different A/B boundaries (i.e., residues 242, 231, and 225, as shown in the bottom of F). (B and C) PRE design. The spin label is at Cys²⁵⁸ in CBD-B. Yellow surfaces are within 25 Å of the spin label. In C-bound (cAMP₂-bound) R_AB, interdomain interactions are minimal (maximal). (D) CBD-A RMSD versus A_onB_on. The closed topology induces the A_off-B_on ➔ A_onB_on transition (black arrow). (E) PRE control. The I_ox/I_red ratios (green circles) track the distance from the spin label (dark green line). Deviations occur in dynamic regions, e.g., flexible linker (gray highlight) (20, 38). (F) As (D), but for CBD-B. (G) I_ox/I_red ratios for the R_AB:C (red), R_AB:C:Rp₂ (orange), and R_AB:Rp₂ (blue) complexes. Dashed lines indicate the average I_ox/I_red ratio for the region with the largest 2QCS (red) versus 1RGS (green) difference (cyan) in distance from the spin label. Solid lines mark the respective average ±1 SD. (H to K) Representative HN TROSY cross-peaks of oxidized and reduced samples. Contour levels were adjusted according to the intensity normalization as per Materials and Methods to account for aggregation.