A Switch In Folding Energy Landscape Governs the Allosteric Activation of Protein Kinase A
England, Jeneffer Patricia
Protein kinases are dynamic molecular switches that are responsible for the regulation of many cellular processes. Mutations, deletions, or gene fusions in protein kinase have been associated with the development of many diseases and has led to the emergence of the protein kinase family as an important therapeutic drug target. In the cell, kinase activity is turned on and off allosterically by intramolecular regulatory domains, flexible linkers, or other interacting proteins. Here, we use single molecule optical tweezers to investigate the mechanism of allosteric regulation of the cAMP-dependent protein kinase A (PKA). The regulatory subunit of PKA harbors two cAMP binding domains (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 catalytic subunit. Moreover, we show that destabilizing mutational effects in one CNB domain efficiently propagate to the other and decreases the folding cooperativity between them. Altogether, our results provide a thermodynamic foundation for the conformational plasticity that protein kinases have in order to adapt and respond to signaling molecules.
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