Mechanism of intracellular cAMP sensor Epac2 activation: cAMP-induced conformational changes identified by amide hydrogen/deuterium exchange mass spectrometry (DXMS)

J Biol Chem. 2011 May 20;286(20):17889-97. doi: 10.1074/jbc.M111.224535. Epub 2011 Mar 17.


Epac2, a guanine nucleotide exchange factor, regulates a wide variety of intracellular processes in response to second messenger cAMP. In this study, we have used peptide amide hydrogen/deuterium exchange mass spectrometry to probe the solution structural and conformational dynamics of full-length Epac2 in the presence and absence of cAMP. The results support a mechanism in which cAMP-induced Epac2 activation is mediated by a major hinge motion centered on the C terminus of the second cAMP binding domain. This conformational change realigns the regulatory components of Epac2 away from the catalytic core, making the later available for effector binding. Furthermore, the interface between the first and second cAMP binding domains is highly dynamic, providing an explanation of how cAMP gains access to the ligand binding sites that, in the crystal structure, are seen to be mutually occluded by the other cAMP binding domain. Moreover, cAMP also induces conformational changes at the ionic latch/hairpin structure, which is directly involved in RAP1 binding. These results suggest that in addition to relieving the steric hindrance imposed upon the catalytic lobe by the regulatory lobe, cAMP may also be an allosteric modulator directly affecting the interaction between Epac2 and RAP1. Finally, cAMP binding also induces significant conformational changes in the dishevelled/Egl/pleckstrin (DEP) domain, a conserved structural motif that, although missing from the active Epac2 crystal structure, is important for Epac subcellular targeting and in vivo functions.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Crystallography, X-Ray
  • Cyclic AMP / chemistry*
  • Cyclic AMP / genetics
  • Cyclic AMP / metabolism
  • Deuterium Exchange Measurement
  • Guanine Nucleotide Exchange Factors / chemistry*
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism
  • Humans
  • Protein Binding
  • Protein Structure, Tertiary
  • Shelterin Complex
  • Telomere-Binding Proteins / chemistry
  • Telomere-Binding Proteins / genetics
  • Telomere-Binding Proteins / metabolism


  • Guanine Nucleotide Exchange Factors
  • RAPGEF4 protein, human
  • Shelterin Complex
  • TERF2IP protein, human
  • Telomere-Binding Proteins
  • Cyclic AMP