title: Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα.
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.
authors.affiliation: Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Madoka
authors.initials: M
authors.lastname: Akimoto
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Eric Tyler
authors.initials: ET
authors.lastname: McNicholl
authors.affiliation: Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Avinash
authors.initials: A
authors.lastname: Ramkissoon
authors.affiliation: Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Kody
authors.initials: K
authors.lastname: Moleschi
authors.affiliation: Department of Chemistry and Biochemistry, Department of Pharmacology and Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California, United States of America.
authors.forename: Susan S
authors.initials: SS
authors.lastname: Taylor
authors.affiliation: Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada.|Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Melacini
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000737:chemistry* / Q000378:metabolism; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004735:Energy Transfer; D004789:Enzyme Activation; D017353:Gene Deletion; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010446:Peptide Fragments; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D020080:Tandem Repeat Sequences
chemical_list: D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D017868:Cyclic AMP-Dependent Protein Kinases; C517758:Prkaca protein, mouse