Fig. 8.

Activation of PKA through selective stabilization of CNB domains. a Unfolding energy landscape of the regulatory subunit in the apo state (left) and bound to cyclic nucleotide (right). The unfolding pathway in the apo state follows a CNB-B-to-CNB-A order in 80% of events (solid blue line). In the other 20%, the apo CNB-A domain unfolds first (dashed light blue line). The apo R241A mutant (dashed red line) has an indistinguishable energy landscape compared with apo wild type. In the presence of cAMP, the unfolding order in wild type is as follows: the N3A motif, the CNB-B domain, and the CNB-A domain. The cAMP-bound R241A mutant follows a similar unfolding order, but the CNB-B domain unfolds with a lower energy barrier and has lower stability due to the lack of stabilizing inter-domain interactions. The cGMP-bound wild type follows an unfolding pathway that is quantitatively and qualitatively different to that of the cAMP state (see main text for details). The height of the energy barriers reflects the folded and unfolded-state lifetimes of the CNB domains (Supplementary Tables 2–5). The energy landscapes have been normalized to the unfolded state. b Activation mechanism of PKA showing intermediate states and their cAMP-initiated interactions. Steps and interactions disrupted by R241A and cGMP are shown as “X” (complete disruption) or “down-arrow” (decreased in magnitude). c Top: interaction network initiated by cAMP binding involves stabilizing (dashed black arrows) and destabilizing (flat black arrow) coupling (top). Effect of R241A (middle) and cGMP (bottom) on interaction network