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J. Biol. Chem., Vol. 283, Issue 10, 6501-6508, March 7, 2008

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Structural Dynamics in the Activation of Epac^*

Shannon M. Harper¹, Hans Wienk¹, Rainer W. Wechselberger, Johannes L. Bos, Rolf Boelens², and Holger Rehmann³

From the Department of Physiological Chemistry and Centre for Biomedical Genetics, University Medical Center Utrecht, 3584 CG Utrecht and NMR Spectroscopy, Bijvoet Center for Biomolecular Research, Utrecht University, 3584 CH Utrecht, The Netherlands

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.

Received for publication, September 19, 2007 , and in revised form, December 6, 2007.

^* This work was supported by a long-term fellowship from the Human Frontier Science Program (to S. M. H.), by the Centre for Biomedical Genetics (to H. W. and R. B.), and the Chemical Section of the Netherlands Organization for Scientific Research (to H. R. and J. L. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed: Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Tel.: 31-30-253-4035; Fax: 31-30-253-7623; E-mail: r.boelens{at}chem.uu.nl.

³ To whom correspondence may be addressed: UMCU Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. Tel.: 31-88-75-68961; Fax: 31-88-75-68101; E-mail: h.rehmann{at}UMCutrecht.nl.

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