Molecular Features of Product Release for the PKA Catalytic Cycle

Although ADP release is the rate limiting step in product turnover by protein kinase A, the steps and motions involved in this process are not well resolved. Here we report the apo and ADP bound structures of the myristylated catalytic subunit of PKA at 2.9 and 3.5 Å resolution, respectively. The ADP bound structure adopts a conformation that does not conform to the previously characterized open, closed, or intermediate states. In the ADP bound structure, the C-terminal tail and Gly-rich loop are more closed than in the open state adopted in the apo structure but are also much more open than the intermediate or closed conformations. Furthermore, ADP binds at the active site with only one magnesium ion, termed Mg2 from previous structures. These structures thus support a model where ADP release proceeds through release of the substrate and Mg1 followed by lifting of the Gly-rich loop and disengagement of the C-terminal tail. Coupling of these two structural elements with the release of the first metal ion fills in a key step in the catalytic cycle that has been missing and supports an ensemble of correlated conformational states that mediate the full catalytic cycle for a protein kinase

Suppressor mutations identified from the <i>prkaca-L173A</i> background.

<p>Suppressor mutations identified from the <i>prkaca-L173A</i> background.</p

Residues highlighted to interact with the nucleotide base in PKA.

<p>When PKA is in an ATP-bound state, the small lobe (gray) and glycine-rich loop move toward the large lobe (sand) to enclose the nucleotide-binding pocket around ATP. <i>A</i>, A global view of PKA in a nucleotide-bound state. Eight residues have been identified to interact with the nucleotide base: Leu 49, Val 57, Val 104, Met 120, Tyr 122, Leu 173, Thr 183, and Phe 327 (highlighted yellow). The bound nucleotide is represented as a ball-and-stick figure. The C-terminal tail is colored red, and PKI is colored magenta. <i>B</i>, Zoomed view into the nucleotide binding pocket. The nucleotide binding pocket is largely preformed with the exception of Val 57, Met 120, Leu 173, and Phe 327 which undergo structural changes upon nucleotide binding. Structure adapted from PDB accession number 1ATP and modeled using Pymol.</p

Suppressor mutations identified from the <i>prkaca-F327A</i> background.

<p>Suppressor mutations identified from the <i>prkaca-F327A</i> background.</p

The L173A mutant protein is stably expressed but inactive, whereas the F327A mutant has lowered protein expression.

<p><i>A</i>, Protein expression of each PKA mutant were measured. Of the two mutants that could not complement synthetic lethality in the <i>tpk</i> null strain, F327A also demonstrated very low protein expression. <i>B</i>, The catalytic activity of each PKA mutant was determined. Both L173A and F327A mutants had nearly undetectable catalytic activity.</p

Suppressor mutations from the <i>prkaca-F327A</i> background.

<p><i>A</i>, Highlighted suppressor mutations identified from the <i>prkaca-F327A</i> background. The original F327A mutation is highlighted in yellow, and suppressor mutations are shown in blue. PKA features are highlighted as follows: small lobe, gray; large lobe, sand; C-terminal tail,red; PKI: magenta. Some frequently highlighted mutations are labeled. The image was made using PDB file 1ATP and modeled using PyMol. <i>B</i>, Mutations isolated from the same suppressor are connected by a dashed line. Single suppressor mutations are listed above the corresponding secondary structural elements of PKA. Secondary structural elements are represented where alpha-helices are red and beta-sheets are blue.</p

Suppressors identified from the <i>prkaca-L173A</i> background.

<p><i>A</i>, The original L173A mutation is highlighted yellow and the suppressor mutations are shown in blue. PKA features are highlighted as follows: small lobe, gray; large lobe, sand; C-terminal tail,red; PKI: magenta. Some frequently isolated suppressor mutations are labeled. Mutations are shown using PDB file 1ATP and modeled using PyMol. <i>B</i>, Multiple mutations isolated from the same suppressor are connected by a dashed line and generally aligned with secondary structural elements of PKA. Secondary structural elements are represented where alpha-helices are red and beta-sheets are blue.</p

Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein Kinase A Using H/D Fractionation Factors

Protein kinase A is a prototypical phosphoryl transferase, sharing its catalytic core (PKA-C) with the entire kinase family. PKA-C substrate recognition, active site organization, and product release depend on the enzyme’s conformational transitions from the open to the closed state, which regulate its allosteric cooperativity. Here, we used equilibrium nuclear magnetic resonance hydrogen/deuterium (H/D) fractionation factors (φ) to probe the changes in the strength of hydrogen bonds within the kinase upon binding the nucleotide and a pseudosubstrate peptide (PKI_5–24). We found that the φ values decrease upon binding both ligands, suggesting that the overall hydrogen bond networks in both the small and large lobes of PKA-C become stronger. However, we observed several important exceptions, with residues displaying higher φ values upon ligand binding. Notably, the changes in φ values are not localized near the ligand binding pockets; rather, they are radiated throughout the entire enzyme. We conclude that, upon ligand and pseudosubstrate binding, the hydrogen bond networks undergo extensive reorganization, revealing that the open-to-closed transitions require global rearrangements of the internal forces that stabilize the enzyme’s fold

Mapping the Free Energy Landscape of PKA Inhibition and Activation: A Double-Conformational Selection Model for the Tandem cAMP-Binding Domains of PKA RIα

<div><p>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.</p></div

Probing CBD-A/B interactions in apo, C-bound, and cAMP₂-bound RIα (91–379) through CBD-B deletion.

<p><b>(A)</b> Overlay of the HN-TROSY spectra of C-bound RIα (91–379) and RIα (91–244), in which CBD-B is deleted. <b>(B)</b> Correlation between the combined chemical shifts (CCS) of C-bound RIα (91–379) and RIα (91–244). <b>(C, D)</b> As in panels (A, B), but for the apo forms of RIα (91–379) and RIα (91–244). <b>(E, F)</b> 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. <b>(G)</b> 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). <b>(H)</b> 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).</p