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, Avinash Ramkissoon, Kody Moleschi, Susan S. Taylor, Giuseppe Melacini

Research output: Contribution to journalArticle

8 Citations (Scopus)

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.

Original languageEnglish
Article numbere1002305
JournalPLoS Biology
Volume13
Issue number11
DOIs
Publication statusPublished - 2015 Nov 30
Externally publishedYes

Fingerprint

cAMP-dependent protein kinase
cyclic AMP
Cyclic AMP-Dependent Protein Kinases
Cyclic AMP
Free energy
Carrier Proteins
Chemical activation
energy
nuclear magnetic resonance spectroscopy
Conservation
Magnetic Resonance Spectroscopy
Nuclear magnetic resonance
Apolipoproteins C
protein subunits
Eukaryota
eukaryotic cells
Conformations
Catalytic Domain
receptors

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

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; Taylor, Susan S.; Melacini, Giuseppe.

In: PLoS Biology, Vol. 13, No. 11, e1002305, 30.11.2015.

Research output: Contribution to journalArticle

Akimoto, Madoka ; McNicholl, Eric Tyler ; Ramkissoon, Avinash ; Moleschi, Kody ; Taylor, Susan S. ; Melacini, Giuseppe. / Mapping the Free Energy Landscape of PKA Inhibition and Activation : A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα. In: PLoS Biology. 2015 ; Vol. 13, No. 11.
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