Limits...
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.

Show MeSH

Related in: MedlinePlus

Intra- and inter-domain RIα dynamics.Dynamics of WT (black closed circles) and W260A (green open circles) RIα (119–379):cAMP2 mapped through residue-specific reduced spectral densities at 0 Hz, ωN, and ωN + ωH frequencies in panels (A–C), respectively. Green vertical arrows in panel (A) mark residues of the helical region between the two β-barrels that are subject to fast H/D exchange (within the dead time) in W260A but not in the WT sample. The reduced spectral densities predicted based on the cAMP2- and C-bound RIα structures in the absence of internal motions are shown in orange and blue, respectively. The PDB codes of the structures used for the reduced spectral densities prediction are reported in the figure. Key regions subject to significant CBD-A versus B differences in dynamic profiles are highlighted in grey and with dashed ovals. The dashed horizontal lines in panels (A, B) indicate the average reduced spectral density values at 0 Hz and at ωN for the WT (black) and W260A (green) constructs.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4664472&req=5

pbio.1002305.g005: Intra- and inter-domain RIα dynamics.Dynamics of WT (black closed circles) and W260A (green open circles) RIα (119–379):cAMP2 mapped through residue-specific reduced spectral densities at 0 Hz, ωN, and ωN + ωH frequencies in panels (A–C), respectively. Green vertical arrows in panel (A) mark residues of the helical region between the two β-barrels that are subject to fast H/D exchange (within the dead time) in W260A but not in the WT sample. The reduced spectral densities predicted based on the cAMP2- and C-bound RIα structures in the absence of internal motions are shown in orange and blue, respectively. The PDB codes of the structures used for the reduced spectral densities prediction are reported in the figure. Key regions subject to significant CBD-A versus B differences in dynamic profiles are highlighted in grey and with dashed ovals. The dashed horizontal lines in panels (A, B) indicate the average reduced spectral density values at 0 Hz and at ωN for the WT (black) and W260A (green) constructs.

Mentions: The W260A mutation causes major chemical shift changes in the presence of excess cAMP (Fig 4A, green bars) and these changes mimic those caused by the deletion of the whole CBD-B (Fig 4B). These chemical shift patterns suggest the single point W260A mutation is sufficient to disrupt the CBD-A/B interface. The disruption of the CBD-A/B interface caused by the W260A mutation was also independently confirmed through 15N-relaxation measurements, which were analyzed in terms of reduced spectral densities (Fig 5). The W260A mutation causes a decrease in the average J(0) value and a concurrent increase in the average J(ωN) value (Fig 5A and 5B, red vertical arrows; Table 1), which are indicative of a reduction of the effective correlation time for overall tumbling, as expected upon de-correlation of the tumbling motions of the two adjacent CBDs, i.e., the W260A mutant conforms better than WT to a model of two domains joined by a flexible linker. This result is independently confirmed by the observation that W260A enhances the flexibility of the helical region connecting the two tandem CBDs, as supported by the fact that several residues in this region experience a reduction of J(0) values (Fig 5A, red dashed oval) and an enhanced solvent exposure (Fig 5A, green arrows) due to the W260A mutation. Hence, the changes in dynamics caused by W260A corroborate the chemical shift perturbation results (Fig 4A and 4B), indicating that the W260A mutation is sufficient to disrupt the CBD-A/B interface.


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)

Intra- and inter-domain RIα dynamics.Dynamics of WT (black closed circles) and W260A (green open circles) RIα (119–379):cAMP2 mapped through residue-specific reduced spectral densities at 0 Hz, ωN, and ωN + ωH frequencies in panels (A–C), respectively. Green vertical arrows in panel (A) mark residues of the helical region between the two β-barrels that are subject to fast H/D exchange (within the dead time) in W260A but not in the WT sample. The reduced spectral densities predicted based on the cAMP2- and C-bound RIα structures in the absence of internal motions are shown in orange and blue, respectively. The PDB codes of the structures used for the reduced spectral densities prediction are reported in the figure. Key regions subject to significant CBD-A versus B differences in dynamic profiles are highlighted in grey and with dashed ovals. The dashed horizontal lines in panels (A, B) indicate the average reduced spectral density values at 0 Hz and at ωN for the WT (black) and W260A (green) constructs.
© Copyright Policy
Related In: Results  -  Collection

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

pbio.1002305.g005: Intra- and inter-domain RIα dynamics.Dynamics of WT (black closed circles) and W260A (green open circles) RIα (119–379):cAMP2 mapped through residue-specific reduced spectral densities at 0 Hz, ωN, and ωN + ωH frequencies in panels (A–C), respectively. Green vertical arrows in panel (A) mark residues of the helical region between the two β-barrels that are subject to fast H/D exchange (within the dead time) in W260A but not in the WT sample. The reduced spectral densities predicted based on the cAMP2- and C-bound RIα structures in the absence of internal motions are shown in orange and blue, respectively. The PDB codes of the structures used for the reduced spectral densities prediction are reported in the figure. Key regions subject to significant CBD-A versus B differences in dynamic profiles are highlighted in grey and with dashed ovals. The dashed horizontal lines in panels (A, B) indicate the average reduced spectral density values at 0 Hz and at ωN for the WT (black) and W260A (green) constructs.
Mentions: The W260A mutation causes major chemical shift changes in the presence of excess cAMP (Fig 4A, green bars) and these changes mimic those caused by the deletion of the whole CBD-B (Fig 4B). These chemical shift patterns suggest the single point W260A mutation is sufficient to disrupt the CBD-A/B interface. The disruption of the CBD-A/B interface caused by the W260A mutation was also independently confirmed through 15N-relaxation measurements, which were analyzed in terms of reduced spectral densities (Fig 5). The W260A mutation causes a decrease in the average J(0) value and a concurrent increase in the average J(ωN) value (Fig 5A and 5B, red vertical arrows; Table 1), which are indicative of a reduction of the effective correlation time for overall tumbling, as expected upon de-correlation of the tumbling motions of the two adjacent CBDs, i.e., the W260A mutant conforms better than WT to a model of two domains joined by a flexible linker. This result is independently confirmed by the observation that W260A enhances the flexibility of the helical region connecting the two tandem CBDs, as supported by the fact that several residues in this region experience a reduction of J(0) values (Fig 5A, red dashed oval) and an enhanced solvent exposure (Fig 5A, green arrows) due to the W260A mutation. Hence, the changes in dynamics caused by W260A corroborate the chemical shift perturbation results (Fig 4A and 4B), indicating that the W260A mutation is sufficient to disrupt the CBD-A/B interface.

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