Abstract
Exchange proteins directly activated by cAMP (EPACs) are guanine nucleotide-exchange factors for the small GTPases Rap1 and Rap2 and represent a key receptor for the ubiquitous cAMP second messenger in eukaryotes. The cAMP-dependent activation of apoEPAC is typically rationalized in terms of a preexisting equilibrium between inactive and active states. Structural and mutagenesis analyses have shown that one of the critical determinants of the EPAC activation equilibrium is a cluster of salt bridges formed between the catalytic core and helices alpha1 and alpha2 at the N terminus of the cAMP binding domain and commonly referred to as ionic latch (IL). The IL stabilizes the inactive states in a closed topology in which access to the catalytic domain is sterically occluded by the regulatory moiety. However, it is currently not fully understood how the IL is allosterically controlled by cAMP. Chemical shift mapping studies consistently indicate that cAMP does not significantly perturb the structure of the IL spanning sites within the regulatory region, pointing to cAMP-dependent dynamic modulations as a key allosteric carrier of the cAMP-signal to the IL sites. Here, we have therefore investigated the dynamic profiles of the EPAC1 cAMP binding domain in its apo, cAMP-bound, and Rp-cAMPS phosphorothioate antagonist-bound forms using several 15N relaxation experiments. Based on the comparative analysis of dynamics in these three states, we have proposed a model of EPAC activation that incorporates the dynamic features allosterically modulated by cAMP and shows that cAMP binding weakens the IL by increasing its entropic penalty due to dynamic enhancements.
Highlights
JULY 11, 2008 VOLUME 283 NUMBER 28 second messenger in mammals (1–3)
Our data indicate that a similar enhancement of ms-s flexibility occurs in exchange protein directly activated by cAMP (EPAC), as indicated by the NMR dispersion (NMRD) dispersions measured for residues Ala-272 and -277 in the EPAC1 phosphate binding cassette (PBC) and Phe-232 and Ile-243 in the EPAC1 2-3 site, which are consistently higher in the Rp-cAMPSbound state relative to the cAMP-bound form (Fig. 6)
Considering that cAMP binding alone does not cause any significant structural rearrangement for ␣1–2, the increase of conformational entropy promoted at this site by cAMP emerges as a key mechanism for a cAMP-dependent weakening of the inhibitory salt bridges mediated by the ionic latch
Summary
JULY 11, 2008 VOLUME 283 NUMBER 28 second messenger in mammals (1–3). The interaction of cAMP with EPAC results in the activation of the guanine-nucleotide exchange in the small GTPases Rap and Rap (1, 2), leading to the cAMP-dependent control of a wide array of critical signaling pathways underlying diverse cellular functions, ranging from insulin secretion to memory enhancement and cell adhesion (4 –10). Two cAMP-dependent EPAC isoforms are currently known (Fig. 1a) Both EPAC1 and -2 are multidomain proteins with an N-terminal regulatory region (RR), including the cAMP binding domains (CBDs) and a C-terminal catalytic region (CR), containing a CDC25-homology module (CDC25HD) that functions as a guanine-nucleotide-exchange factor (GEF) (Fig. 1a). A deletion mutant (i.e. EPAC2⌬306) in which the IL is weakened through the removal of one of the CR/RR salt bridges displays a striking 5-fold increase in the maximum exchange activity (kmax) (16), indicating that the integral IL contributes to shifting the EPAC equilibrium toward the inactive state It is currently not clear how the IL sites located in the N-terminal helical bundle are controlled by cAMP, which docks in the distal PBC and BBR, embedded within the -subdomain. We have investigated primarily by classical NMR 15N relaxation experiments as well as by multi-offset NMR dispersion measurements the EPAC1h-(149 –318) construct in its apo-, cAMP-bound (holo), and Rp-cAMPS-bound states
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