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Conformational landscape of the protein kinase A Inhibitor PKI studied by fluorescence and NMR spectroscopy

Cyclic AMP‐Dependent protein kinase A (PKA) is a ubiquitous phosphoryl transferase that participates in numerous cellular signaling pathway. Inside the cell, the activity and localization of PKA are finely regulated by endogenous inhibitor proteins, such as the heat‐stable protein kinase A inhibitor (PKI). PKI plays a key role also in the nuclear regulation and cytoplasmic translocation of the catalytic subunit of PKA (PKA‐C). To understand the molecular mechanism underlying recognition of PKA‐C by PKI, we characterized the change in the conformational landscape of PKIα upon binding PKA‐C as well as the mechanism of binding, using solution NMR and fluorescence spectroscopy. We demonstrated that PKI free in solution has minimal secondary structural elements with only two transient helices and has transient intramolecular interaction between the C‐ and N‐terminus. When a ternary complex between PKA‐C, PKI and a non‐hydrolysable ATP mimic (ATPγN) is formed, only the portion of the peptide that directly interacts with the PKA‐C undergoes significant structural rearrangement, while the majority of the peptide is still unfolded. However, our experiments also indicated that binding to PKA‐C confers allosteric changes toward the nuclear export signal (NES) region present on PKI. Also our transient kinetics data reveal that the mechanism involves at least two distinct populations of PKA‐C binding to PKI with subsequent structural rearrangement and the flux through these competing pathways depend on ligand concentration. These studies not only elucidate the structural and mechanistic basis of PKA‐C:PKI interaction, but also represent the basis for further investigation on the role of the PKA‐C:PKI interaction toward regulation and nuclear export.