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. 2018 Aug 7;115(32):E7478-E7485.
doi: 10.1073/pnas.1802510115. Epub 2018 Jul 23.

Switching of the Folding-Energy Landscape Governs the Allosteric Activation of Protein Kinase A

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Free PMC article

Switching of the Folding-Energy Landscape Governs the Allosteric Activation of Protein Kinase A

Jeneffer P England et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Protein kinases are dynamic molecular switches that sample multiple conformational states. The regulatory subunit of PKA harbors two cAMP-binding domains [cyclic nucleotide-binding (CNB) domains] that oscillate between inactive and active conformations dependent on cAMP binding. The cooperative binding of cAMP to the CNB domains activates an allosteric interaction network that enables PKA to progress from the inactive to active conformation, unleashing the activity of the catalytic subunit. Despite its importance in the regulation of many biological processes, the molecular mechanism responsible for the observed cooperativity during the activation of PKA remains unclear. Here, we use optical tweezers to probe the folding cooperativity and energetics of domain communication between the cAMP-binding domains in the apo state and bound to the catalytic subunit. Our study provides direct evidence of a switch in the folding-energy landscape of the two CNB domains from energetically independent in the apo state to highly cooperative and energetically coupled in the presence of the catalytic subunit. Moreover, we show that destabilizing mutational effects in one CNB domain efficiently propagate to the other and decrease the folding cooperativity between them. Taken together, our results provide a thermodynamic foundation for the conformational plasticity that enables protein kinases to adapt and respond to signaling molecules.

Keywords: allostery; cAMP; kinase; optical tweezers; single molecule.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structure, domain organization, and optical tweezers assay to study PKA. (A) Domain organization of PKA. The catalytic subunit has an N- and a C-lobe that form an active-site cleft. The regulatory subunit has a modular domain organization. The D/D (shown in grey) and the flexible linker domain (residues 71–110, shown in orange) contain the IS that mimics the peptide substrate of the catalytic subunit. Two CNB domains, CNB-A (light purple) and CNB-B (dark purple), are connected by the αB/C helix (maroon). The N3A motif of the CNB-A domain is shown in teal. The cAMP-binding pocket of each CNB domain is shown in yellow. (B, Left) The PKA complex is shown in the inactive form in which the catalytic subunit (tan) is bound to the regulatory subunit (light and dark purple). (Right) The binding of two molecules of cAMP results in a conformational change in the regulatory subunit (cAMP-bound form) and enables the release of the active catalytic subunit (boxed in green). (C) Optical tweezers experimental set-up. The regulatory subunit is tethered between two polystyrene beads by the attachment of DNA handles at positions flanking the CNB domains (Y120/S376) shown in cyan in B). The PKA complex is formed in trans with the catalytic subunit, ATP, and Mg2+ present in the microfluidic chamber.
Fig. 2.
Fig. 2.
Mechanical unfolding of the wild-type regulatory subunit. (A) Representative force–extension curves for the mechanical unfolding of the regulatory subunit (residues 71–379) in the absence (Left) and presence (Right) of the catalytic subunit. In the absence of the catalytic subunit, only one unfolding pathway is observed with two rips. In the presence of the catalytic subunit, two unfolding pathways, I and II, are observed that correspond to different conformations of the regulatory subunit. (B) Titration of the catalytic subunit increases the percentage of unfolding pathway I [labeled “% Tightly Bound” (TB)]. Saturation occurs at 100 nM catalytic subunit. (Inset) The same percentage of unfolding pathway I is observed at 100 nM catalytic subunit with 10-s and 20-s refolding times at 1 pN. (C) Unfolding (Unf) and refolding (Ref) force probability distributions for the CNB-A domain are shown for the apo state and bound conformations (light and dark purple bars, respectively). An increase in the unfolding force of 3 pN is observed in the presence of the catalytic subunit. (Inset) Refolding force probability distribution in the presence of the catalytic subunit (white bars). (D) Folded-state lifetimes (τ0) as a function of force for the CNB-A domain in the absence and presence of the catalytic subunit (light and dark purple squares, respectively). White squares show the unfolded-state lifetimes (τU) as a function of force for the CNB-A domain in the presence of the catalytic subunit. (E) Unfolding and refolding force probability distributions for the CNB-B domain in the apo state and bound conformations (light and dark blue bars, respectively). The presence of the catalytic subunit stabilizes the CNB-B domain by ∼6 pN. (Inset) Refolding force probability distribution in the presence of the catalytic subunit. (F) Folded-state lifetimes (τF) as a function of force for the CNB-B domain in the absence and presence of the catalytic subunit (light and dark blue squares, respectively). Light yellow squares show the unfolded-state lifetimes (τU) as a function of force for the CNB-A domain in the presence of the catalytic subunit. The lines in D and F correspond to the best fit of SI Appendix, Eq. S2, and the lines in C and E correspond to the best fit of SI Appendix, Eq. S3 (SI Appendix, Section 1.7).
Fig. 3.
Fig. 3.
Mechanical unfolding of the R333K regulatory subunit. (A) Representative force–extension curves for the mechanical unfolding of the R333K mutant regulatory subunit in the absence and presence of the catalytic subunit. In the absence of the catalytic subunit, or apo state, we observed two unfolding rips at 5 pN and 13 pN. The rip occurring at 13 pN has a change in extension that is comparable to the CNB-A domain. In contrast, the rip seen at 5 pN has a much lower extension change than that of the CNB-B domain, indicating that this mutant protein in the apo state is partially folded. Incubation with the catalytic subunit facilitates and recovers the correct folding of the R333K mutant regulatory subunit. Unfolding occurs in two unfolding pathways, I and II, a behavior similar to the wild-type construct. (B) Unfolding (Unf) force probability distributions for the R333K mutant and wild-type CNB-B domain bound to the catalytic subunit (dark red and dark blue bars, respectively). (Inset) Refolding (Ref) force probability distribution of the R333K mutant CNB-B domain in the presence of the catalytic subunit (light orange bars). (C) Lifetimes (τ0) as a function of force for the CNB-B domain in the presence of the catalytic subunit. Light orange and dark red spheres correspond to unfolded (τU) and folded-state (τF) lifetimes for the R333K mutant. Light yellow and dark blue squares correspond to unfolded and folded state lifetimes for the wild-type protein. (D) Unfolding (Unf) force probability distributions for the R333K mutant and wild-type CNB-A domain bound to the catalytic subunit (light brown and dark purple bars, respectively). No refolding transitions were observed for the R333K mutant CNB-A domain in the presence of the catalytic subunit. (E) Folded-state lifetimes (τF) as a function of force for the R333K mutant and wild-type CNB-A domain in the absence and presence of the catalytic subunit (light brown spheres and dark purple squares, respectively). The lines in C and E correspond to the best fit of SI Appendix, Eq. S2, and the lines in B and D correspond to the best fit of SI Appendix, Eq. S3 (SI Appendix, Section 1.7).
Fig. 4.
Fig. 4.
A switch in the folding-energy landscape of the CNB domains controls the activation of PKA. (Upper) An ensemble is observed for the PKA complex in the inactive state (green box), in which the CNB-B domain exists in a tightly and a loosely bound conformation. The R333K mutation shifts the inactive ensemble toward a greater fraction of the loosely bound conformation (tan box). In the unfolding pathway, the CNB-B domain unfolds first because a greater degree of surface contacts exists between the CNB-A domain and the catalytic subunit via the N3A motif (shown in teal) and the αB/C helix (shown in maroon). The unfolding of the CNB-B domain results in the loss of the Arg241(R):Asp267(R):Arg194(C) salt bridge (Enlarged View) (41), which exposes a weaker unfolding point, or Achilles heel unfolding point, and the fast dissociation of the CNB-A domain from the catalytic subunit. (Lower) This cooperative unfolding pathway is also observed in the energetics of the unfolding free-energy landscape. In the absence of the catalytic subunit (apo state, red trace), a lower energy barrier is required to unfold the CNB-B domain (21 kBT) than the CNB-A domain (37 kBT). In contrast, in the tightly bound conformation (blue trace) a higher energy barrier is required to unfold the CNB-B domain (34 kBT) than the CNB-A domain (18 kBT). This change in energy barriers results in highly cooperative unfolding behavior between the two CNB domains bound to the catalytic subunit. A destabilizing mutation in the CNB-B domain (R333K, dashed green trace) partially decouples the cooperativity between the two CNB domains bound to the catalytic subunit. The energy difference in the folded state (XNative) is not to scale to better distinguish the differences between the energy barriers for the apo, wild-type, and R333K protein constructs.

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