Elucidating the Mechanism of Recognition and Binding of Protein Kinase Inhibitor by Protein Kinase a using NMR and Fluorescence Spectroscopy

Abstract

Cyclic AMP-dependent protein kinase A (PKA) mediates many crucial cellular events including gene expression, metabolism, and cardiac contractility by catalyzing phosphoryl transfer. The endogenous thermostable protein kinase inhibitor (PKI) is a key protein that regulates the activity and intracellular localization of PKA through its two functional motifs: the kinase inhibitory domain and nuclear export signal domain. The kinase inhibitory domain binds specifically with the catalytic subunit of PKA (PKA-C) with high affinity while the nuclear export signal is responsible for shuttling the kinase out of the nucleus. PKI is intrinsically disordered in its free form but adopts residual lowly populated structure through the two distinct domains. Upon PKA-C binding, PKI folds into a more ordered conformation with chemical shift changes propagated throughout the inhibitory domain, however the remainder of the inhibitor is disordered. Here, we aim to elucidate the mechanism by which PKA recognizes and binds PKI by a combination of NMR and fluorescence spectroscopy. Our findings reveal the molecular mechanism of interaction that will help us understand how an intrinsically disordered protein can perform its dual regulatory functions. Our future work will focus on the structural basis of nuclear export of PKA-C upon PKI binding, revealing the mechanistic details of regulation of PKA outside of cAMP control.