The functional response of a signaling system to an allosteric stimulus often depends on subcellular conditions, a phenomenon known as pluripotent allostery. For example, a single allosteric modulator, Rp-cAMPS, of the prototypical protein kinase A (PKA) switches from antagonist to agonist depending on MgATP levels. However, the mechanism underlying such pluripotent allostery has remained elusive for decades. Using nuclear magnetic resonance spectroscopy, ensemble models, kinase assays, and molecular dynamics simulations, we show that allosteric pluripotency arises from surprisingly divergent responses of highly homologous tandem domains. The differential responses perturb domain-domain interactions and remodel the free-energy landscape of inhibitory excited states sampled by the regulatory subunit of PKA. The resulting activation threshold values are comparable to the effective free energy of regulatory and catalytic subunit binding, which depends on metabolites, substrates, and mutations, explaining pluripotent allostery and warranting a general redefinition of allosteric targets to include specific subcellular environments.

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.

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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...