Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα

Protein kinase a (pka) is the major receptor for the cyclic adenosine monophosphate (camp) secondary messenger in eukaryotes. camp binds to two tandem camp-binding domains (cbd-a and -b) within the regulatory subunit of pka (r),unleashing the activity of the catalytic subunit (c). while cbd-a in riα is required for pka inhibition and activation,cbd-b functions as a “gatekeeper” domain that modulates the control exerted by cbd-a. preliminary evidence suggests that cbd-b dynamics are critical for its gatekeeper function. to test this hypothesis,here we investigate by nuclear magnetic resonance (nmr) the two-domain construct riα (91–379) in its apo,camp2,and c-bound forms. our comparative nmr analyses lead to a double conformational selection model in which each apo cbd dynamically samples both active and inactive states independently of the adjacent cbd within a nearly degenerate free energy landscape. such degeneracy is critical to explain the sensitivity of cbd-b to weak interactions with c and its high affinity for camp. binding of camp eliminates this degeneracy,as it selectively stabilizes the active conformation within each cbd and inter-cbd contacts,which require both camp and w260. the latter is contributed by cbd-b and mediates capping of the camp bound to cbd-a. the inter-cbd interface is dispensable for intra-cbd conformational selection,but is indispensable for full activation of pka as it occludes c-subunit recognition sites within cbd-a. in addition,the two structurally homologous camp-bound cbds exhibit marked differences in their residual dynamics profiles,supporting the notion that conservation of structure does not necessarily imply conservation of dynamics. © 2015 akimoto et al.