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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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Conformational selection in CBD-A and -B of RIα (91–379).(A, B) Overlay of apo, cAMP2-bound, and C-bound RIα (91–379) for cross-peaks of representative residues sensing the active versus inactive equilibria in CBD-A (A) and CBD-B (B). (C) Fractional inhibition of apo RIα (91–379) (X) relative to cAMP2- and C-bound RIα (91–379), assuming they represent the active and inactive forms of PKA, respectively. The inset illustrates how X was measured using the CHEmical Shift Projection Analysis (CHESPA) method. The bar graph shows the average X values observed for residues in CBD-A and -B exhibiting a linear pattern (/cosθ/ > 0.95). (D) Alternative evaluation of the fractional inhibition of apo RIα (91–379) (X) through the slope of the (δapo−δcAMP) versus (δC−δcAMP) plot, where δapo, δcAMP, and δC are the chemical shifts of apo, cAMP2- and C-bound RIα (91–379). Closed and open circles indicate 1H and 15N chemical shifts, respectively. This plot was restricted to CBD-A residues that are sufficiently removed from the cAMP-dependent interfaces (e.g., R:C, R:cAMP and CBD-A:CBD-B) to report primarily on the active versus inactive equilibrium of CBD-A. (E) Similar to panel (D) but for CBD-B.
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pbio.1002305.g002: Conformational selection in CBD-A and -B of RIα (91–379).(A, B) Overlay of apo, cAMP2-bound, and C-bound RIα (91–379) for cross-peaks of representative residues sensing the active versus inactive equilibria in CBD-A (A) and CBD-B (B). (C) Fractional inhibition of apo RIα (91–379) (X) relative to cAMP2- and C-bound RIα (91–379), assuming they represent the active and inactive forms of PKA, respectively. The inset illustrates how X was measured using the CHEmical Shift Projection Analysis (CHESPA) method. The bar graph shows the average X values observed for residues in CBD-A and -B exhibiting a linear pattern (/cosθ/ > 0.95). (D) Alternative evaluation of the fractional inhibition of apo RIα (91–379) (X) through the slope of the (δapo−δcAMP) versus (δC−δcAMP) plot, where δapo, δcAMP, and δC are the chemical shifts of apo, cAMP2- and C-bound RIα (91–379). Closed and open circles indicate 1H and 15N chemical shifts, respectively. This plot was restricted to CBD-A residues that are sufficiently removed from the cAMP-dependent interfaces (e.g., R:C, R:cAMP and CBD-A:CBD-B) to report primarily on the active versus inactive equilibrium of CBD-A. (E) Similar to panel (D) but for CBD-B.

Mentions: As a first step towards mapping the functional conformational equilibria of apo PKA RIα (91–379), we compared it to two reference states, i.e., C-bound RIα (91–379), which is assumed to trap the inhibitory (“inactive”) conformation of RIα (91–379), and cAMP2-bound RIα (91–379), which is expected to represent the uninhibited (“active”) form of PKA-R. The comparative spectral analysis of apo versus C- versus cAMP2-bound RIα (91–379) is illustrated in Fig 2. Panels 2A and 2B focus on two representative residues of CBD-A and -B, i.e., G223 and S297, respectively, which are sufficiently removed from the cAMP and C-subunit binding interfaces to report primarily on the conformational equilibria of RIα (91–379). For both residues, the cross-peaks arising from the apo, C, and cAMP2-bound forms define a clear linear pattern, which is indicative of apo RIα (91–379) sampling two states (i.e., the active and inactive conformations) through an exchange that is fast in the chemical shift NMR time scale at the field of data acquisition (i.e., 700 MHz).


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)

Conformational selection in CBD-A and -B of RIα (91–379).(A, B) Overlay of apo, cAMP2-bound, and C-bound RIα (91–379) for cross-peaks of representative residues sensing the active versus inactive equilibria in CBD-A (A) and CBD-B (B). (C) Fractional inhibition of apo RIα (91–379) (X) relative to cAMP2- and C-bound RIα (91–379), assuming they represent the active and inactive forms of PKA, respectively. The inset illustrates how X was measured using the CHEmical Shift Projection Analysis (CHESPA) method. The bar graph shows the average X values observed for residues in CBD-A and -B exhibiting a linear pattern (/cosθ/ > 0.95). (D) Alternative evaluation of the fractional inhibition of apo RIα (91–379) (X) through the slope of the (δapo−δcAMP) versus (δC−δcAMP) plot, where δapo, δcAMP, and δC are the chemical shifts of apo, cAMP2- and C-bound RIα (91–379). Closed and open circles indicate 1H and 15N chemical shifts, respectively. This plot was restricted to CBD-A residues that are sufficiently removed from the cAMP-dependent interfaces (e.g., R:C, R:cAMP and CBD-A:CBD-B) to report primarily on the active versus inactive equilibrium of CBD-A. (E) Similar to panel (D) but for CBD-B.
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pbio.1002305.g002: Conformational selection in CBD-A and -B of RIα (91–379).(A, B) Overlay of apo, cAMP2-bound, and C-bound RIα (91–379) for cross-peaks of representative residues sensing the active versus inactive equilibria in CBD-A (A) and CBD-B (B). (C) Fractional inhibition of apo RIα (91–379) (X) relative to cAMP2- and C-bound RIα (91–379), assuming they represent the active and inactive forms of PKA, respectively. The inset illustrates how X was measured using the CHEmical Shift Projection Analysis (CHESPA) method. The bar graph shows the average X values observed for residues in CBD-A and -B exhibiting a linear pattern (/cosθ/ > 0.95). (D) Alternative evaluation of the fractional inhibition of apo RIα (91–379) (X) through the slope of the (δapo−δcAMP) versus (δC−δcAMP) plot, where δapo, δcAMP, and δC are the chemical shifts of apo, cAMP2- and C-bound RIα (91–379). Closed and open circles indicate 1H and 15N chemical shifts, respectively. This plot was restricted to CBD-A residues that are sufficiently removed from the cAMP-dependent interfaces (e.g., R:C, R:cAMP and CBD-A:CBD-B) to report primarily on the active versus inactive equilibrium of CBD-A. (E) Similar to panel (D) but for CBD-B.
Mentions: As a first step towards mapping the functional conformational equilibria of apo PKA RIα (91–379), we compared it to two reference states, i.e., C-bound RIα (91–379), which is assumed to trap the inhibitory (“inactive”) conformation of RIα (91–379), and cAMP2-bound RIα (91–379), which is expected to represent the uninhibited (“active”) form of PKA-R. The comparative spectral analysis of apo versus C- versus cAMP2-bound RIα (91–379) is illustrated in Fig 2. Panels 2A and 2B focus on two representative residues of CBD-A and -B, i.e., G223 and S297, respectively, which are sufficiently removed from the cAMP and C-subunit binding interfaces to report primarily on the conformational equilibria of RIα (91–379). For both residues, the cross-peaks arising from the apo, C, and cAMP2-bound forms define a clear linear pattern, which is indicative of apo RIα (91–379) sampling two states (i.e., the active and inactive conformations) through an exchange that is fast in the chemical shift NMR time scale at the field of data acquisition (i.e., 700 MHz).

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