Journal Article

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

Akimoto, Madoka, McNicholl, Eric Tyler, Ramkissoon, Avinash, Moleschi, Kody, Melacini, Giuseppe, Taylor, Susan S.

2015

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Amino Acid Substitution / Animals / Binding Sites / Cattle / Cyclic adenylic acid / Cyclic AMP - chemistry / Cyclic AMP - metabolism / Cyclic AMP-Dependent Protein Kinase Catalytic Subunits - chemistry / Cyclic AMP-Dependent Protein Kinase Catalytic Subunits - genetics / Cyclic AMP-Dependent Protein Kinase Catalytic Subunits - metabolism / Cyclic AMP-Dependent Protein Kinase RIalpha Subunit - chemistry / Cyclic AMP-Dependent Protein Kinase RIalpha Subunit - genetics / Cyclic AMP-Dependent Protein Kinase RIalpha Subunit - metabolism / Cyclic AMP-Dependent Protein Kinases - chemistry / Cyclic AMP-Dependent Protein Kinases - genetics / Cyclic AMP-Dependent Protein Kinases - metabolism / Energy Transfer / Enzyme Activation / Gene Deletion / Mice / Models, Molecular / Mutation / Peptide Fragments / Physiological aspects / Protein Conformation / Protein Interaction Domains and Motifs / Protein Interaction Mapping / Protein kinases / Recombinant Fusion Proteins - chemistry / Recombinant Fusion Proteins - metabolism / Tandem Repeat Sequences