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.
Bibliographical noteFunding Information:
This work was supported by the National Institute of Health GM 100310 to G.V and GM46736
the University of Utah was funded by the Department of Energy (Dr. J. Trewhella).
© 2020, eLife Sciences Publications Ltd. All rights reserved.
- Binding Kinetics
- Coupled Binding and Folding
- Intrinsically Disordered Proteins
- Protein Kinase Inhibitor
- Protein kinases
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, U.S. Gov't, Non-P.H.S.
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Analysis of the kinetics of binding of Protein Kinase A Inhibitor alpha (PKIa) to cAMP-dependent protein kinase a catalytic subunit (PKA-C)