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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Allosteric Regulation / physiology
  • Catalytic Domain / genetics
  • Catalytic Domain / physiology*
  • Cyclic AMP / chemistry
  • Cyclic AMP / metabolism*
  • Cyclic AMP-Dependent Protein Kinases / chemistry
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Enzyme Assays
  • Molecular Dynamics Simulation
  • Mutation
  • Optical Tweezers
  • Protein Binding / physiology
  • Protein Domains / physiology
  • Protein Folding*
  • Signal Transduction / physiology*


  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases