Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A.

Cristina Olivieri,Jiali Gao,Joseph M Muretta,Benjamin R Stultz,Jonggul Kim,Geoffrey C Li,David Dt Thomas,Matthew Neibergall,Yingjie Wang,Gianluigi Veglia,Fernando Porcelli,Donald K Blumenthal,Manu V S,Susan S Taylor

doi:10.7554/elife.55607

Abstract

In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI. After a fast binding step in which PKA-C selects PKI's most competent conformations, PKI folds upon binding through a slow conformational rearrangement within the enzyme's binding pocket. The high-affinity and pseudo-substrate regions of PKI become more structured and the transient interactions with the kinase augment the helical content of the nuclear export sequence, which is then poised to recruit the CRM1/RanGTP complex for nuclear translocation. The multistate binding mechanism featured by PKA-C/PKI complex represents a paradigm on how disordered, ancillary proteins (or protein domains) are able to operate multiple functions such as inhibiting the kinase while recruiting other regulatory proteins for nuclear export.

Highlights

The cAMP-dependent protein kinase A (PKA) is a ubiquitous phosphoryl transferase that regulates numerous cellular signaling pathways (Taylor et al, 2012)
Using a combination of NMR spectroscopy, small angle X-ray scattering (SAXS), stopped-flow fluorescence, replica-averaged metadynamics (RAM), and Markov model analysis, here we provide a comprehensive view of the molecular mechanism for recognition of protein kinase Inhibitor alpha (PKIa) by protein kinase a catalytic subunit (PKA-C)
We propose that the multistate pathway for PKIa recognition by PKA-C is preparatory for the recruitment by CRM1/ RanGTP for nuclear export and regulation of gene expression

Summary

Introduction

The cAMP-dependent protein kinase A (PKA) is a ubiquitous phosphoryl transferase that regulates numerous cellular signaling pathways (Taylor et al, 2012). As for other eukaryotic protein kinases, PKA regulatome comprises globular domains as well as extensive disordered regions, which are essential for protein-protein interactions and signaling (Akimoto et al, 2013; Gogl et al, 2019; Pellicena and Kuriyan, 2006). The activity and localization of PKA-C within different cellular compartments are finely regulated by ancillary proteins such as the regulatory subunits (R), A-kinaseanchoring proteins (AKAPs) (Bauman and Scott, 2002; Johnson and Lewis, 2001), and the heat-stable protein kinase A inhibitor (PKI) (Dalton and Dewey, 2006). While R-subunits are primarily responsible for the regulation and localization of PKA-C in the cytoplasm through interaction with

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Conclusion