Journal Article

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

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

2020

Overview

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.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Active Transport, Cell Nucleus / Animals / Binding sites / Cyclic AMP-Dependent Protein Kinases - genetics / Cyclic AMP-Dependent Protein Kinases - metabolism / Cytoplasm / Enzyme inhibitors / Enzymes / Escherichia coli / Experiments / Exportin 1 Protein / Gene expression / Intracellular Signaling Peptides and Proteins - genetics / Intracellular Signaling Peptides and Proteins - metabolism / isotopic labeling / Karyopherins - genetics / Karyopherins - metabolism / Kinases / Localization / Magnetic Resonance Spectroscopy / Markov Chains / Mice / NMR / Nuclear magnetic resonance / Nuclear transport / protein expression / Protein kinase A / Protein kinase inhibitors / Protein Kinase Inhibitors - metabolism / Proteins / Rabbits / Receptors, Cytoplasmic and Nuclear - genetics / Receptors, Cytoplasmic and Nuclear - metabolism / recombinant dna / Recruitment / Regulatory proteins / Spectrum analysis / Standard deviation / Structural Biology and Molecular Biophysics