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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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(A) “Double Conformational Selection” Model proposed for PKA-RIα activation. The inactive and active states of each CBD are represented through ovals and rectangles, respectively. The grey horizontal bar indicates that free energy differences are minimal (i.e., of the order of ~RT). Dotted lines denote interactions with the C-subunit of PKA. cAMP is shown as solid black circles. RAB refers to the PKA-RIα region spanning the two tandem cAMP-binding domains (CBD-A and -B). The cAMP recognition motifs for both CBDs are reported as BBR:cAMP:hinge-lid, where BBR stands for base-binding region. Orange (red) denotes enhanced ms-μs (ps-ns) dynamics. See text in the Discussion for a full explanation. (B) Inter-domain contact profile based on the structure of cAMP2-bound RIα (91–379) (PDB Code: 1RGS) and SASA changes occurring upon deletion of the 113–232 region (for contacts in the 233–376 segment) or the 233–376 region (for contacts in the 113–232 segment). The boundary between the two deleted regions (i.e., residues 232–233) is based on the observation that the 233–244 region is flexible in RIα (91–244):cAMP (46). (C) Profile of RIα (91–379):C contacts as mapped by SASA variations upon deletion of the C-subunit from the structure of the RIα (91–379) R333K:C complex (PDB Code: 2QCS). CBD-A regions experiencing SASA changes in both panels (B) and (C) (i.e., CBD-A residues involved in both CBD-A/B contacts as well as interactions with the C-subunit) are highlighted by dashed boxes.
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pbio.1002305.g006: (A) “Double Conformational Selection” Model proposed for PKA-RIα activation. The inactive and active states of each CBD are represented through ovals and rectangles, respectively. The grey horizontal bar indicates that free energy differences are minimal (i.e., of the order of ~RT). Dotted lines denote interactions with the C-subunit of PKA. cAMP is shown as solid black circles. RAB refers to the PKA-RIα region spanning the two tandem cAMP-binding domains (CBD-A and -B). The cAMP recognition motifs for both CBDs are reported as BBR:cAMP:hinge-lid, where BBR stands for base-binding region. Orange (red) denotes enhanced ms-μs (ps-ns) dynamics. See text in the Discussion for a full explanation. (B) Inter-domain contact profile based on the structure of cAMP2-bound RIα (91–379) (PDB Code: 1RGS) and SASA changes occurring upon deletion of the 113–232 region (for contacts in the 233–376 segment) or the 233–376 region (for contacts in the 113–232 segment). The boundary between the two deleted regions (i.e., residues 232–233) is based on the observation that the 233–244 region is flexible in RIα (91–244):cAMP (46). (C) Profile of RIα (91–379):C contacts as mapped by SASA variations upon deletion of the C-subunit from the structure of the RIα (91–379) R333K:C complex (PDB Code: 2QCS). CBD-A regions experiencing SASA changes in both panels (B) and (C) (i.e., CBD-A residues involved in both CBD-A/B contacts as well as interactions with the C-subunit) are highlighted by dashed boxes.

Mentions: The comparative NMR analyses of the dynamic conformational equilibria of CBD-A and -B presented here support a “double conformational selection” model for the cAMP-dependent activation of PKA RIα (Fig 6A). In the absence of cAMP the PKA-RIα region spanning the two tandem cAMP-binding domains (CBD-A and -B), denoted as RAB, samples four states populating a nearly degenerate free-energy landscape, in which each CBD accesses both C-binding competent (“inactive”) and cAMP-binding incompetent (“active”) conformations with comparable populations, as shown in Fig 2. In the absence of cAMP, such active versus inactive sampling of each CBD occurs independently of the adjacent CBD, due to the absence of significant inter-domain interactions, as shown in Fig 3A–3D. Out of these four nearly degenerate states, the “inactive-inactive” state exhibits the highest affinity for the C-subunit, as both domains are primed for binding C and the inter-domain helical region is also available for interacting with C. The CBD-B affinity for the C-subunit is known to be lower than that of CBD-A, but weak interactions between CBD-B and the C-subunit are still sufficient to drive the conformational equilibria towards the inactive state due to the apo RAB near degeneracy.


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)

(A) “Double Conformational Selection” Model proposed for PKA-RIα activation. The inactive and active states of each CBD are represented through ovals and rectangles, respectively. The grey horizontal bar indicates that free energy differences are minimal (i.e., of the order of ~RT). Dotted lines denote interactions with the C-subunit of PKA. cAMP is shown as solid black circles. RAB refers to the PKA-RIα region spanning the two tandem cAMP-binding domains (CBD-A and -B). The cAMP recognition motifs for both CBDs are reported as BBR:cAMP:hinge-lid, where BBR stands for base-binding region. Orange (red) denotes enhanced ms-μs (ps-ns) dynamics. See text in the Discussion for a full explanation. (B) Inter-domain contact profile based on the structure of cAMP2-bound RIα (91–379) (PDB Code: 1RGS) and SASA changes occurring upon deletion of the 113–232 region (for contacts in the 233–376 segment) or the 233–376 region (for contacts in the 113–232 segment). The boundary between the two deleted regions (i.e., residues 232–233) is based on the observation that the 233–244 region is flexible in RIα (91–244):cAMP (46). (C) Profile of RIα (91–379):C contacts as mapped by SASA variations upon deletion of the C-subunit from the structure of the RIα (91–379) R333K:C complex (PDB Code: 2QCS). CBD-A regions experiencing SASA changes in both panels (B) and (C) (i.e., CBD-A residues involved in both CBD-A/B contacts as well as interactions with the C-subunit) are highlighted by dashed boxes.
© Copyright Policy
Related In: Results  -  Collection

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

pbio.1002305.g006: (A) “Double Conformational Selection” Model proposed for PKA-RIα activation. The inactive and active states of each CBD are represented through ovals and rectangles, respectively. The grey horizontal bar indicates that free energy differences are minimal (i.e., of the order of ~RT). Dotted lines denote interactions with the C-subunit of PKA. cAMP is shown as solid black circles. RAB refers to the PKA-RIα region spanning the two tandem cAMP-binding domains (CBD-A and -B). The cAMP recognition motifs for both CBDs are reported as BBR:cAMP:hinge-lid, where BBR stands for base-binding region. Orange (red) denotes enhanced ms-μs (ps-ns) dynamics. See text in the Discussion for a full explanation. (B) Inter-domain contact profile based on the structure of cAMP2-bound RIα (91–379) (PDB Code: 1RGS) and SASA changes occurring upon deletion of the 113–232 region (for contacts in the 233–376 segment) or the 233–376 region (for contacts in the 113–232 segment). The boundary between the two deleted regions (i.e., residues 232–233) is based on the observation that the 233–244 region is flexible in RIα (91–244):cAMP (46). (C) Profile of RIα (91–379):C contacts as mapped by SASA variations upon deletion of the C-subunit from the structure of the RIα (91–379) R333K:C complex (PDB Code: 2QCS). CBD-A regions experiencing SASA changes in both panels (B) and (C) (i.e., CBD-A residues involved in both CBD-A/B contacts as well as interactions with the C-subunit) are highlighted by dashed boxes.
Mentions: The comparative NMR analyses of the dynamic conformational equilibria of CBD-A and -B presented here support a “double conformational selection” model for the cAMP-dependent activation of PKA RIα (Fig 6A). In the absence of cAMP the PKA-RIα region spanning the two tandem cAMP-binding domains (CBD-A and -B), denoted as RAB, samples four states populating a nearly degenerate free-energy landscape, in which each CBD accesses both C-binding competent (“inactive”) and cAMP-binding incompetent (“active”) conformations with comparable populations, as shown in Fig 2. In the absence of cAMP, such active versus inactive sampling of each CBD occurs independently of the adjacent CBD, due to the absence of significant inter-domain interactions, as shown in Fig 3A–3D. Out of these four nearly degenerate states, the “inactive-inactive” state exhibits the highest affinity for the C-subunit, as both domains are primed for binding C and the inter-domain helical region is also available for interacting with C. The CBD-B affinity for the C-subunit is known to be lower than that of CBD-A, but weak interactions between CBD-B and the C-subunit are still sufficient to drive the conformational equilibria towards the inactive state due to the apo RAB near degeneracy.

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