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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 M, McNicholl ET, Ramkissoon A, Moleschi K, Taylor SS, Melacini G - PLoS Biol. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada.

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.

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Construct design and architecture of PKA RIα.(A) Domain organization of the RIα subunit of PKA and its functional constructs that ensure cAMP-dependent inhibition of the catalytic subunit (C). (B) Binding equilibria of PKA RIα. Even when apo RIα is a transient low-population intermediate, it is still a critical thermodynamic determinant of the cAMP-dependent regulation of PKA. (C) Structures of PKA RIα (91–379) bound to either cAMP (grey, PDB code 1RGS) or the C-subunit (orange, PDB code 2QCS for the R333K mutant of RIα (91–379)). The two structures are superimposed through the β-barrel of cAMP-binding domain (CBD) A. Selected regions and residues are labeled. The arrow shows the change in the position of CBD-B relative to CBD-A occurring upon cAMP-binding.
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pbio.1002305.g001: Construct design and architecture of PKA RIα.(A) Domain organization of the RIα subunit of PKA and its functional constructs that ensure cAMP-dependent inhibition of the catalytic subunit (C). (B) Binding equilibria of PKA RIα. Even when apo RIα is a transient low-population intermediate, it is still a critical thermodynamic determinant of the cAMP-dependent regulation of PKA. (C) Structures of PKA RIα (91–379) bound to either cAMP (grey, PDB code 1RGS) or the C-subunit (orange, PDB code 2QCS for the R333K mutant of RIα (91–379)). The two structures are superimposed through the β-barrel of cAMP-binding domain (CBD) A. Selected regions and residues are labeled. The arrow shows the change in the position of CBD-B relative to CBD-A occurring upon cAMP-binding.

Mentions: Cyclic adenosine monophosphate (cAMP) is an ancient secondary messenger, and in higher eukaryotes, Protein Kinase A (PKA) is the major receptor for cAMP. The cAMP-dependent activation of PKA is utilized by a wide variety of extracellular stimuli to control the respective intra-cellular responses, such as regulation of the immune system and cell proliferation [1–4]. In the resting state, PKA exists as an inhibited tetramer formed by a dimeric regulatory subunit (R2), with each R-protomer binding and inhibiting one equivalent of catalytic subunit (C). Upon binding of four equivalents of cAMP to R2, the R2C2 tetramer at least partially dissociates, releasing the active C-subunit to phosphorylate downstream substrates [5–10]. The R-subunit of PKA is a multi-domain protein (Fig 1A), starting from a dimerization-docking domain followed by a flexible linker, which includes an inhibitory site for C and is in turn followed by two tandem cAMP-binding domains (CBD-A and -B) that provide additional contact sites for binding the C-subunit [11,12]. 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 C-inhibition and cAMP-dependent activation of full-length PKA [13–15]. The structures of this construct have been solved in both the cAMP2-bound (i.e., active wild type [WT]) [14] and C-bound forms (i.e., inactive R333K mutant) [13], revealing major conformational changes that underlie the cAMP-dependent activation of PKA (Fig 1C). Furthermore, the dynamics of apo CBD-A have been recently shown to be another central determinant of the allosteric control of PKA activation by cAMP [16]. However, less is known about the dynamics of CBD-B and how they relate to the physiological function of this domain.


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 M, McNicholl ET, Ramkissoon A, Moleschi K, Taylor SS, Melacini G - PLoS Biol. (2015)

Construct design and architecture of PKA RIα.(A) Domain organization of the RIα subunit of PKA and its functional constructs that ensure cAMP-dependent inhibition of the catalytic subunit (C). (B) Binding equilibria of PKA RIα. Even when apo RIα is a transient low-population intermediate, it is still a critical thermodynamic determinant of the cAMP-dependent regulation of PKA. (C) Structures of PKA RIα (91–379) bound to either cAMP (grey, PDB code 1RGS) or the C-subunit (orange, PDB code 2QCS for the R333K mutant of RIα (91–379)). The two structures are superimposed through the β-barrel of cAMP-binding domain (CBD) A. Selected regions and residues are labeled. The arrow shows the change in the position of CBD-B relative to CBD-A occurring upon cAMP-binding.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664472&req=5

pbio.1002305.g001: Construct design and architecture of PKA RIα.(A) Domain organization of the RIα subunit of PKA and its functional constructs that ensure cAMP-dependent inhibition of the catalytic subunit (C). (B) Binding equilibria of PKA RIα. Even when apo RIα is a transient low-population intermediate, it is still a critical thermodynamic determinant of the cAMP-dependent regulation of PKA. (C) Structures of PKA RIα (91–379) bound to either cAMP (grey, PDB code 1RGS) or the C-subunit (orange, PDB code 2QCS for the R333K mutant of RIα (91–379)). The two structures are superimposed through the β-barrel of cAMP-binding domain (CBD) A. Selected regions and residues are labeled. The arrow shows the change in the position of CBD-B relative to CBD-A occurring upon cAMP-binding.
Mentions: Cyclic adenosine monophosphate (cAMP) is an ancient secondary messenger, and in higher eukaryotes, Protein Kinase A (PKA) is the major receptor for cAMP. The cAMP-dependent activation of PKA is utilized by a wide variety of extracellular stimuli to control the respective intra-cellular responses, such as regulation of the immune system and cell proliferation [1–4]. In the resting state, PKA exists as an inhibited tetramer formed by a dimeric regulatory subunit (R2), with each R-protomer binding and inhibiting one equivalent of catalytic subunit (C). Upon binding of four equivalents of cAMP to R2, the R2C2 tetramer at least partially dissociates, releasing the active C-subunit to phosphorylate downstream substrates [5–10]. The R-subunit of PKA is a multi-domain protein (Fig 1A), starting from a dimerization-docking domain followed by a flexible linker, which includes an inhibitory site for C and is in turn followed by two tandem cAMP-binding domains (CBD-A and -B) that provide additional contact sites for binding the C-subunit [11,12]. 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 C-inhibition and cAMP-dependent activation of full-length PKA [13–15]. The structures of this construct have been solved in both the cAMP2-bound (i.e., active wild type [WT]) [14] and C-bound forms (i.e., inactive R333K mutant) [13], revealing major conformational changes that underlie the cAMP-dependent activation of PKA (Fig 1C). Furthermore, the dynamics of apo CBD-A have been recently shown to be another central determinant of the allosteric control of PKA activation by cAMP [16]. However, less is known about the dynamics of CBD-B and how they relate to the physiological function of this domain.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada.

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.

Show MeSH
Related in: MedlinePlus