Understanding cAMP-dependent allostery by NMR spectroscopy: Comparative analysis of the EPAC1 cAMP-binding domain in its Apo and cAMP-bound states
Department of Chemistry and Chemical Biology, McMaster University, Hamilton, Ontario, Canada Journal of the American Chemical Society
(Impact Factor: 12.11).
12/2007; 129(46):14482-92. DOI: 10.1021/ja0753703
cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a significantly more extended allosteric network that, unlike PKA, involves a tight coupling between the alpha- and beta-subdomains of the EPAC CBD. The proposed mechanism of allosteric activation will serve as a basis to understand agonism and antagonism in the EPAC system and provides also a general paradigm for how small ligands control protein-protein interfaces.
Available from: PubMed Central
- "However, the maximal P o at 3 mM cGMP for Construct 3 (0.73 ± 0.15; n = 3) is far higher Substitution of the fCNGA2 PB cassette increases pro-action efficacy We selected Construct 3 for a more detailed comparison with the previously characterized Construct 2, because these two constructs differ only in five residues within the key PB cassette region. In multiple cyclic nucleotide– activated proteins, movement of the PB cassette in response to ligand binding is believed to be essential for propagating conformational change to other parts of the BD (Canaves and Taylor, 2002; Rehmann et al., 2003; Clayton et al., 2004; Kim et al., 2005; Mazhab-Jafari et al., 2007). Thus, although we knew that the five The P o parameter traditionally plotted in dose– response curves does not bear a simple relation with the underlying energetics of gate opening; the more pertinent parameter is the equilibrium constant for the poregating reaction ([open]/[closed], in a simple model of than for Construct 2 (0.18 ± 0.05; n = 3), so that the entire cGMP dose–response curves for the two channels are dramatically separated. "
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ABSTRACT: Cyclic nucleotide-gated (CNG) channels bind cGMP or cAMP in a cytoplasmic ligand-binding domain (BD), and this binding typically increases channel open probability (P(o)) without inducing desensitization. However, the catfish CNGA2 (fCNGA2) subtype exhibits bimodal agonism, whereby steady-state P(o) increases with initial cGMP-binding events ("pro" action) up to a maximum of 0.4, but decreases with subsequent cGMP-binding events ("con" action) occurring at concentrations >3 mM. We sought to clarify if low pro-action efficacy was either necessary or sufficient for con action to operate. To find BD residues responsible for con action or low pro-action efficacy or both, we constructed chimeric CNG channels: subregions of the fCNGA2 BD were substituted with corresponding sequence from the rat CNGA4 BD, which does not support con action. Constructs were expressed in frog oocytes and tested by patch clamp of cell-free membranes. For nearly all BD elements, we found at least one construct where replacing that element preserved robust con action, with a ratio of steady-state conductances, g((10 mM cGMP))/g((3 mM cGMP)) < 0.75. When all of the BD sequence C terminal of strand β6 was replaced, g((10 mM cGMP))/g((3 mM cGMP)) was increased to 0.95 ± 0.05 (n = 7). However, this apparent attenuation of con action could be explained by an increase in the efficacy of pro action for all agonists, controlled by a conserved "phosphate-binding cassette" motif that contacts ligand; this produces high P(o) values that are less sensitive to shifts in gating equilibrium. In contrast, substituting a single valine in the N-terminal helix αA abolished con action (g((30 mM cGMP))/g((3 mM cGMP)) increased to 1.26 ± 0.24; n = 7) without large increases in pro-action efficacy. Our work dissociates the two functional features of low pro-action efficacy and con action, and moreover identifies a separate structural determinant for each.
Available from: Stephen J Yarwood
- "In the absence of cAMP the proteins favour the closed auto-inhibited conformation , while cAMP binding shifts the equilibrium towards the active conformation. X-ray crystallography has provided highly informative snapshots of the open (Rehmann et al., 2008) and closed (Rehmann et al., 2006) conformations of EPAC2 while NMR spectroscopy has been used to explore how ligand binding affects the dynamics of the critical conformational switch in EPAC1 (Harper et al., 2007; Mazhab-Jafari et al., 2007; Das et al., 2008). Lower resolution techniques have also been applied to the problem (Yu et al., 2006; Brock et al., 2007), but their limitations, that is, the requirement for an adequate prior model, are revealed in the light of the structure of the EPAC2·Sp-CAMPS·RAP1B complex (Rehmann et al., 2008). "
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ABSTRACT: It has now been over 10 years since efforts to completely understand the signalling actions of cAMP (3'-5'-cyclic adenosine monophosphate) led to the discovery of exchange protein directly activated by cAMP (EPAC) proteins. In the current review we will highlight important advances in the understanding of EPAC structure and function and demonstrate that EPAC proteins mediate multiple actions of cAMP in cells, revealing future targets for pharmaceutical intervention. It has been known for some time that drugs that elevate intracellular cAMP levels have proven therapeutic benefit for diseases ranging from depression to inflammation. The challenge now is to determine which of these positive actions of cAMP involve activation of EPAC-regulated signal transduction pathways. EPACs are specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, which they activate independently of the classical routes for cAMP signalling, cyclic nucleotide-gated ion channels and protein kinase A. Rather, EPAC activation is triggered by internal conformational changes induced by direct interaction with cAMP. Leading from this has been the development of EPAC-specific agonists, which has helped to delineate numerous cellular actions of cAMP that rely on subsequent activation of EPAC. These include regulation of exocytosis and the control of cell adhesion, growth, division and differentiation. Recent work also implicates EPAC in the regulation of anti-inflammatory signalling in the vascular endothelium, namely negative regulation of pro-inflammatory cytokine signalling and positive support of barrier function. Further elucidation of these important signalling mechanisms will no doubt support the development of the next generation of anti-inflammatory drugs.
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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.
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