Abstract
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.