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Proc Natl Acad Sci U S A. 2018 Aug 7;115(32):E7478-E7485. doi: 10.1073/pnas.1802510115. Epub 2018 Jul 23.

Switching of the folding-energy landscape governs the allosteric activation of protein kinase A.

Author information

1
Department of Chemistry, Georgetown University, Washington, DC 20057.
2
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030.
3
Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX 77030.
4
Dan L Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030.
5
Center for Cancer Epigenetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030.
6
Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093; staylor@ucsd.edu rodrigo.maillard@georgetown.edu.
7
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093.
8
Department of Chemistry, Georgetown University, Washington, DC 20057; staylor@ucsd.edu rodrigo.maillard@georgetown.edu.

Abstract

Protein kinases are dynamic molecular switches that sample multiple conformational states. The regulatory subunit of PKA harbors two cAMP-binding domains [cyclic nucleotide-binding (CNB) domains] that oscillate between inactive and active conformations dependent on cAMP binding. The cooperative binding of cAMP to the CNB domains activates an allosteric interaction network that enables PKA to progress from the inactive to active conformation, unleashing the activity of the catalytic subunit. Despite its importance in the regulation of many biological processes, the molecular mechanism responsible for the observed cooperativity during the activation of PKA remains unclear. Here, we use optical tweezers to probe the folding cooperativity and energetics of domain communication between the cAMP-binding domains in the apo state and bound to the catalytic subunit. Our study provides direct evidence of a switch in the folding-energy landscape of the two CNB domains from energetically independent in the apo state to highly cooperative and energetically coupled in the presence of the catalytic subunit. Moreover, we show that destabilizing mutational effects in one CNB domain efficiently propagate to the other and decrease the folding cooperativity between them. Taken together, our results provide a thermodynamic foundation for the conformational plasticity that enables protein kinases to adapt and respond to signaling molecules.

KEYWORDS:

allostery; cAMP; kinase; optical tweezers; single molecule

PMID:
30038016
PMCID:
PMC6094112
[Available on 2019-02-07]
DOI:
10.1073/pnas.1802510115
[Indexed for MEDLINE]

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