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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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Probing CBD-A/B interactions in apo, C-bound, and cAMP2-bound RIα (91–379) through CBD-B deletion.(A) Overlay of the HN-TROSY spectra of C-bound RIα (91–379) and RIα (91–244), in which CBD-B is deleted. (B) Correlation between the combined chemical shifts (CCS) of C-bound RIα (91–379) and RIα (91–244). (C, D) As in panels (A, B), but for the apo forms of RIα (91–379) and RIα (91–244). (E, F) As in panels (A, B), but for the cAMP2-bound form of RIα (91–379) and the cAMP-bound form of RIα (91–244). Color codes for panels A, C, and E are indicated in the figure and representative CBD-A and -B cross-peaks are labeled. (G) Map of above-average RIα (91–379):cAMP2 versus RIα (91–244):cAMP CCS differences for residues <226 (blue spheres) onto the structure of RIα (91–379):cAMP2 (PDB Code: 1RGS). (H) Cyan spheres represent CBD-A residues experiencing solvent accessible surface area (SASA) changes upon deletion of residues 226–379, which are highlighted with a grey surface. This SASA map was built using the same structure as in panel (G).
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pbio.1002305.g003: Probing CBD-A/B interactions in apo, C-bound, and cAMP2-bound RIα (91–379) through CBD-B deletion.(A) Overlay of the HN-TROSY spectra of C-bound RIα (91–379) and RIα (91–244), in which CBD-B is deleted. (B) Correlation between the combined chemical shifts (CCS) of C-bound RIα (91–379) and RIα (91–244). (C, D) As in panels (A, B), but for the apo forms of RIα (91–379) and RIα (91–244). (E, F) As in panels (A, B), but for the cAMP2-bound form of RIα (91–379) and the cAMP-bound form of RIα (91–244). Color codes for panels A, C, and E are indicated in the figure and representative CBD-A and -B cross-peaks are labeled. (G) Map of above-average RIα (91–379):cAMP2 versus RIα (91–244):cAMP CCS differences for residues <226 (blue spheres) onto the structure of RIα (91–379):cAMP2 (PDB Code: 1RGS). (H) Cyan spheres represent CBD-A residues experiencing solvent accessible surface area (SASA) changes upon deletion of residues 226–379, which are highlighted with a grey surface. This SASA map was built using the same structure as in panel (G).

Mentions: It should be noted that even if we knew the exact value for the fractional inactivation of CBD-B, the data in Fig 2 alone would not be sufficient to gauge the degree of correlation between the active–inactive conformational transitions in the two tandem domains. For example, if the actual fractional inactivation of each CBD is 50%, two markedly different distributions of state populations are still possible: (1) 50% of active-active state, 50% of inactive-inactive state, and no mixed (inactive-active and active-inactive) states, or (2) 25% of each possible state. In the two scenarios the degree of correlation between the active-inactive transitions in the two domains is clearly different, but in both cases the fractional inactivation for each CBD is 50%. Hence, it is clear that understanding how the inactive versus active intra-CBD equilibria are coupled to each other requires an independent assessment of the CBD-A/B interactions in the context of a combinatorial analysis of CBD states (i.e., active-active, inactive-active, active-inactive, and inactive-inactive CBD-CBD states). For this purpose, we dissected the state-specificity of inter-domain interactions by probing the effect of CBD-B deletion on CBD-A, i.e., by comparing the RIα (91–379) versus RIα (91–244) constructs (Fig 3).


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)

Probing CBD-A/B interactions in apo, C-bound, and cAMP2-bound RIα (91–379) through CBD-B deletion.(A) Overlay of the HN-TROSY spectra of C-bound RIα (91–379) and RIα (91–244), in which CBD-B is deleted. (B) Correlation between the combined chemical shifts (CCS) of C-bound RIα (91–379) and RIα (91–244). (C, D) As in panels (A, B), but for the apo forms of RIα (91–379) and RIα (91–244). (E, F) As in panels (A, B), but for the cAMP2-bound form of RIα (91–379) and the cAMP-bound form of RIα (91–244). Color codes for panels A, C, and E are indicated in the figure and representative CBD-A and -B cross-peaks are labeled. (G) Map of above-average RIα (91–379):cAMP2 versus RIα (91–244):cAMP CCS differences for residues <226 (blue spheres) onto the structure of RIα (91–379):cAMP2 (PDB Code: 1RGS). (H) Cyan spheres represent CBD-A residues experiencing solvent accessible surface area (SASA) changes upon deletion of residues 226–379, which are highlighted with a grey surface. This SASA map was built using the same structure as in panel (G).
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Related In: Results  -  Collection

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pbio.1002305.g003: Probing CBD-A/B interactions in apo, C-bound, and cAMP2-bound RIα (91–379) through CBD-B deletion.(A) Overlay of the HN-TROSY spectra of C-bound RIα (91–379) and RIα (91–244), in which CBD-B is deleted. (B) Correlation between the combined chemical shifts (CCS) of C-bound RIα (91–379) and RIα (91–244). (C, D) As in panels (A, B), but for the apo forms of RIα (91–379) and RIα (91–244). (E, F) As in panels (A, B), but for the cAMP2-bound form of RIα (91–379) and the cAMP-bound form of RIα (91–244). Color codes for panels A, C, and E are indicated in the figure and representative CBD-A and -B cross-peaks are labeled. (G) Map of above-average RIα (91–379):cAMP2 versus RIα (91–244):cAMP CCS differences for residues <226 (blue spheres) onto the structure of RIα (91–379):cAMP2 (PDB Code: 1RGS). (H) Cyan spheres represent CBD-A residues experiencing solvent accessible surface area (SASA) changes upon deletion of residues 226–379, which are highlighted with a grey surface. This SASA map was built using the same structure as in panel (G).
Mentions: It should be noted that even if we knew the exact value for the fractional inactivation of CBD-B, the data in Fig 2 alone would not be sufficient to gauge the degree of correlation between the active–inactive conformational transitions in the two tandem domains. For example, if the actual fractional inactivation of each CBD is 50%, two markedly different distributions of state populations are still possible: (1) 50% of active-active state, 50% of inactive-inactive state, and no mixed (inactive-active and active-inactive) states, or (2) 25% of each possible state. In the two scenarios the degree of correlation between the active-inactive transitions in the two domains is clearly different, but in both cases the fractional inactivation for each CBD is 50%. Hence, it is clear that understanding how the inactive versus active intra-CBD equilibria are coupled to each other requires an independent assessment of the CBD-A/B interactions in the context of a combinatorial analysis of CBD states (i.e., active-active, inactive-active, active-inactive, and inactive-inactive CBD-CBD states). For this purpose, we dissected the state-specificity of inter-domain interactions by probing the effect of CBD-B deletion on CBD-A, i.e., by comparing the RIα (91–379) versus RIα (91–244) constructs (Fig 3).

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