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

Madoka Akimoto,Eric Tyler Mcnicholl,Susan S Taylor,Kody Moleschi,Avinash Ramkissoon,Giuseppe Melacini,Ann Stock

doi:10.1371/journal.pbio.1002305

Abstract

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.

Highlights

Cyclic adenosine monophosphate is an ancient secondary messenger, and in higher eukaryotes, Protein Kinase A (PKA) is the major receptor for cAMP
While CBD-A is required for C-subunit inhibition and activation, in RIα CBD-B functions as a “gatekeeper” domain that modulates the control exerted by CBD-A
We comparatively analyzed by Nuclear Magnetic Resonance (NMR) a two-domain construct of the regulatory subunit RIα with no ligand, with cAMP2 bound, and the C-bound form

Summary

Introduction

Cyclic adenosine monophosphate (cAMP) is an ancient secondary messenger, and in higher eukaryotes, Protein Kinase A (PKA) is the major receptor for cAMP. The RIα (91–379) construct spans the inhibitory site and both CBDs (Fig 1A) and recapitulates most of the features that are associated with both Cinhibition and cAMP-dependent activation of full-length PKA [13,14,15]. The structures of this construct have been solved in both the cAMP2-bound (i.e., active wild type [WT]) [14] and Cbound forms (i.e., inactive R333K mutant) [13], revealing major conformational changes that underlie the cAMP-dependent activation of PKA (Fig 1C). Less is known about the dynamics of CBD-B and how they relate to the physiological function of this domain

Methods

Results

Discussion

Conclusion