Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations.

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function.

Divergent allostery reveals critical differences between structurally homologous regulatory domains of Plasmodium falciparum and human protein kinase G.

Malaria is a life-threatening infectious disease primarily caused by the Plasmodium falciparum parasite. The increasing resistance to current antimalarial drugs and their side effects has led to an urgent need for novel malaria drug targets, such as the P. falciparum cGMP-dependent protein kinase (pfPKG). However, PKG plays an essential regulatory role also in the human host. Human cGMP-dependent protein kinase (hPKG) and pfPKG are controlled by structurally homologous cGMP-binding domains (CBDs). Here, we show that despite the structural similarities between the essential CBDs in pfPKG and hPKG, their respective allosteric networks differ significantly. Through comparative analyses of chemical shift covariance analyses, molecular dynamics simulations, and backbone internal dynamics measurements, we found that conserved allosteric elements within the essential CBDs are wired differently in pfPKG and hPKG to implement cGMP-dependent kinase activation. Such pfPKG versus hPKG rewiring of allosteric networks was unexpected because of the structural similarity between the two essential CBDs. Yet, such finding provides crucial information on which elements to target for selective inhibition of pfPKG versus hPKG, which may potentially reduce undesired side effects in malaria treatments.

Allosteric pluripotency: challenges and opportunities.

Allosteric pluripotency arises when the functional response of an allosteric receptor to an allosteric stimulus depends on additional allosteric modulators. Here, we discuss allosteric pluripotency as observed in the prototypical Protein Kinase A (PKA) as well as in other signaling systems, from typical multidomain signaling proteins to bacterial enzymes. We identify key drivers of pluripotent allostery and illustrate how hypothesizing allosteric pluripotency may solve apparent discrepancies currently present in the literature regarding the dual nature of known allosteric modulators. We also outline the implications of allosteric pluripotency for cellular signaling and allosteric drug design, and analyze the challenges and opportunities opened by the pluripotent nature of allostery.

Mechanism of allosteric inhibition in the Plasmodium falciparum cGMP-dependent protein kinase.

Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum Its life cycle is regulated by a cGMP-dependent protein kinase (PfPKG), whose inhibition is a promising antimalaria strategy. Allosteric kinase inhibitors, such as cGMP analogs, offer enhanced selectivity relative to competitive kinase inhibitors. However, the mechanisms underlying allosteric PfPKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective PfPKG antagonist. Using comparative NMR analyses of a key regulatory domain, PfD, in its apo, cGMP-bound, and cGMP analog-bound states, we elucidated its inhibition mechanism of action. Using NMR chemical shift analyses, molecular dynamics simulations, and site-directed mutagenesis, we show that 8-NBD-cGMP inhibits PfPKG not simply by reverting a two-state active versus inactive equilibrium, but by sampling also a distinct inactive "mixed" intermediate. Surface plasmon resonance indicates that the ability to stabilize a mixed intermediate provides a means to effectively inhibit PfPKG, without losing affinity for the cGMP analog. Our proposed model may facilitate the rational design of PfPKG-selective inhibitors for improved management of malaria.

NMR methods to dissect the molecular mechanisms of disease-related mutations (DRMs): Understanding how DRMs remodel functional free energy landscapes.

Elucidating the molecular mechanism of disease-related mutations (DRMs) is a critical first step towards understanding the etiology of genetic disorders. DRMs often modulate biological function by altering the free-energy landscape (FEL) of the protein associated with the mutated gene. FELs typically include ground, as well as excited, yet accessible and functionally relevant, states and DRMs may perturb both the thermodynamics and kinetics of the ground vs. excited and apo vs. holo transitions. NMR is ideally suited to map at atomic-resolution these DRM-induced FEL perturbations. Here, we discuss NMR methods that can elucidate how DRMs remodel regulatory FELs by focusing on a simple, but prototypical, four-state allosteric FEL model. The approaches include the CHEmical Shift Projection Analysis, NMR spin relaxation measurements, and NMR measurements of effector-binding thermodynamics and kinetics. Together, these complementary NMR measurements provide a valuable picture of how DRMs modulate distinct FEL attributes that are critical for dissecting the molecular mechanisms underlying pathological phenotypes.

State-selective frustration as a key driver of allosteric pluripotency.

Allosteric pluripotency arises when an allosteric effector switches from agonist to antagonist depending on the experimental conditions. For example, the Rp-cAMPS ligand of Protein Kinase A (PKA) switches from agonist to antagonist as the MgATP concentration increases and/or the kinase substrate affinity or concentration decreases. Understanding allosteric pluripotency is essential to design effective allosteric therapeutics with minimal side effects. Allosteric pluripotency of PKA arises from divergent allosteric responses of two homologous tandem cAMP-binding domains, resulting in a free energy landscape for the Rp-cAMPS-bound PKA regulatory subunit R1a in which the ground state is kinase inhibition-incompetent and the kinase inhibition-competent state is excited. The magnitude of the free energy difference between the ground non-inhibitory and excited inhibitory states (ΔG R,Gap) relative to the effective free energy of R1a binding to the catalytic subunit of PKA (ΔG R:C) dictates whether the antagonism-to-agonism switch occurs. However, the key drivers of ΔG R,Gap are not fully understood. Here, by analyzing an R1a mutant that selectively silences allosteric pluripotency, we show that a major determinant of ΔG R,Gap unexpectedly arises from state-selective frustration in the ground inhibition-incompetent state of Rp-cAMPS-bound R1a. Such frustration is caused by steric clashes between the phosphate-binding cassette and the helices preceding the lid, which interact with the phosphate and base of Rp-cAMPS, respectively. These clashes are absent in the excited inhibitory state, thus reducing the ΔG R,Gap to values comparable to ΔG R:C, as needed for allosteric pluripotency to occur. The resulting model of allosteric pluripotency is anticipated to assist the design of effective allosteric modulators.

Backbone resonance assignment of the cAMP-binding domains of the protein kinase A regulatory subunit Iα.

Protein kinase A (PKA) is the main receptor for the universal cAMP second messenger. PKA is a tetramer with two catalytic (C) and two regulatory (R) subunits, each including two tandem cAMP-binding domains, i.e. CBD-A and -B. Activation of the complex occurs with cAMP binding first to CBD-B, followed by a second molecule of cAMP binding to CBD-A, which causes the release of the active C-subunit. Unlike previous constructs for eukaryotic cAMP-binding domains (CBDs), the 29.5 kDa construct analyzed here [i.e. RIα (119-379)] spans the CBDs in full and provides insight into inter-domain communication. In this note we report the 1H, 13C, and 15 N backbone assignments of cAMP-bound RIα (119-379) CBDs (BMRB No. 50920).

Dynamical Basis of Allosteric Activation for the Plasmodium falciparum Protein Kinase G.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is required for the progression of the Plasmodium's life cycle and is therefore a promising malaria drug target. PfPKG includes four cGMP-binding domains (CBD-A to CBD-D). CBD-D plays a crucial role in PfPKG regulation as it is the primary determinant for the inhibition and cGMP-dependent activation of the catalytic domain. Hence, it is critical to understand how CBD-D is allosterically regulated by cGMP. Although the apo versus holo conformational changes of CBD-D have been reported, information on the intermediates of the activation pathway is currently lacking. Here, we employed molecular dynamics simulations to model four key states along the thermodynamic cycle for the cGMP-dependent activation of the PfPKG CBD-D domain. The simulations were compared to NMR data, and they revealed that the PfPKG CBD-D activation pathway samples a compact intermediate in which the N- and C-terminal helices approach the central ß-barrel. In addition, by comparing the cGMP-bound active and inactive states, the essential binding interactions that differentiate these states were identified. The identification of structural and dynamical features unique to the cGMP-bound inactive state provides a promising basis to design PfPKG-selective allosteric inhibitors as a viable treatment for malaria.

Allosteric inhibition explained through conformational ensembles sampling distinct "mixed" states.

Allosteric modulation provides an effective avenue for selective and potent enzyme inhibition. Here, we summarize and critically discuss recent advances on the mechanisms of allosteric partial agonists for three representative signalling enzymes activated by cyclic nucleotides: the cAMP-dependent protein kinase (PKA), the cGMP-dependent protein kinase (PKG), and the exchange protein activated by cAMP (EPAC). The comparative analysis of partial agonism in PKA, PKG and EPAC reveals a common emerging theme, i.e. the sampling of distinct "mixed" conformational states, either within a single domain or between distinct domains. Here, we show how such "mixed" states play a crucial role in explaining the observed functional response, i.e. partial agonism and allosteric pluripotency, as well as in maximizing inhibition while minimizing potency losses. In addition, by combining Nuclear Magnetic Resonance (NMR), Molecular Dynamics (MD) simulations and Ensemble Allosteric Modeling (EAM), we also show how to map the free-energy landscape of conformational ensembles containing "mixed" states. By discussing selected case studies, we illustrate how MD simulations and EAM complement NMR to quantitatively relate protein dynamics to function. The resulting NMR- and MD-based EAMs are anticipated to inform not only the design of new generations of highly selective allosteric inhibitors, but also the choice of multidrug combinations.