Structural dynamics in the activation of Epac.
Epac1 is a cAMP-responsive exchange factor for the small G-protein Rap. It consists of a regulatory region containing a cyclic nucleotide binding (CNB) domain and a catalytic region that activates Rap. In the absence of cAMP, access of Rap to the catalytic site is blocked by the regulatory region. We analyzed the conformational states of the CNB domain in the absence and in the presence of cAMP and cAMP analogues by NMR spectroscopy, resulting in the first direct insights into the activation mechanism of Epac. We prove that the CNB domain exists in equilibrium between the inactive and the active conformation, which is shifted by binding of cAMP. cAMP binding results in conformational changes in both the ligand binding pocket and the outer helical segments. We used two different cAMP antagonists that block these successive changes to elucidate the steps of this process. Highlighting the role of dynamics, the superactivator 8-pCPT-2'-O-Me-cAMP induces similar conformational changes as cAMP but causes different internal mobility. The results reveal the critical elements of the CNB domain of Epac required for activation and highlight the role of dynamics in this process.
Harper
Shannon M
SM
Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands.
Wienk
Hans
H
Wechselberger
Rainer W
RW
Bos
Johannes L
JL
Boelens
Rolf
R
Rehmann
Holger
H
D000818:Animals; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary / Q000502:physiology; D000089804:Shelterin Complex; D034501:Telomere-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D008024:Ligands; C116122:RAPGEF3 protein, human; C442864:RAPGEF4 protein, human; D000089804:Shelterin Complex; C440809:TERF2IP protein, human; D034501:Telomere-Binding Proteins; D000242:Cyclic AMP