Fig. 3. Conformational ensembles accessible to apo and C-bound R_AB as a function of Rp-cAMPS binding.

(A) States accessible within the degenerate free-energy landscape of apo R_AB (20). Red triangles and green rectangles denote off and on conformations of each CBD, respectively. The on/off notation refers to the inability/ability of each CBD to promote inhibitory interactions of R with C. (B) States accessible to R_AB:Rp. No interdomain interactions occur when CBD-A is apo. (C) States accessible to R_AB:Rp₂. The dashed lines indicate that the A_off-B_on excited open state sampled by R_AB:Rp₂ is also sampled by C:R_AB:Rp₂ and is a transient intermediate along the cAMP-dependent activation pathway. (D and E) Rp binds first preferentially to CBD-A of C-bound R_AB further stabilizing the inhibitory conformation of CBD-A. (F) States accessible to the quaternary complex C:R_AB:Rp₂, showing that when CBD-B is bound to Rp, it samples the “on” conformation as well. (G to I) Traditional sequential binding of cAMP to C:R_AB, resulting in PKA activation. Unlike the case of Rp binding to C:R_AB, cAMP binds first to CBD-B and then CBD-A (18). The dashed lines illustrate that the inhibition-incompetent R_AB:cAMP₂ complex samples the same ground states as R_AB:Rp₂ as well as one of the excited states.