Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states.
ABSTRACT 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.
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ABSTRACT: Protein kinase A (PKA) is the main receptor for the universal cAMP second messenger. PKA is a tetramer with two catalytic (C) and two regulatory (R) subunits, each including two tandem cAMP binding domains, i.e. CBD-A and -B. Structural investigations of RIalpha have revealed that although CBD-A plays a pivotal role in the cAMP-dependent inhibition of C, the main function of CBD-B is to regulate the access of cAMP to site A. To further understand the mechanism underlying the cross-talk between CBD-A and -B, we report here the NMR investigation of a construct of R, RIalpha-(119-379), which unlike previous fragments characterized by NMR, spans in full both CBDs. Our NMR studies were also extended to two mutants, R209K and the corresponding R333K, which severely reduce the affinity of cAMP for CBD-A and -B, respectively. The comparative NMR analysis of wild-type RIalpha-(119-379) and of the two domain silencing mutations has led to the definition at an unprecedented level of detail of both intra- and interdomain allosteric networks, revealing several striking differences between the two CBDs. First, the two domains, although homologous in sequence and structure, exhibit remarkably different responses to the R/K mutations especially at the beta2-3 allosteric "hot spot." Second, although the two CBDs are reciprocally coupled at the level of local unfolding of the hinge, the A-to-B and B-to-A pathways are dramatically asymmetrical at the level of global unfolding. Such an asymmetric interdomain cross-talk ensures efficiency and robustness in both the activation and de-activation of PKA.Journal of Biological Chemistry 03/2010; 285(20):15523-37. · 4.65 Impact Factor
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ABSTRACT: The exchange protein directly activated by cAMP (EPAC) is a key receptor of cAMP in eukaryotes and controls critical signaling pathways. Currently, no residue resolution information is available on the full-length EPAC dynamics, which are known to be pivotal determinants of allostery. In addition, no information is presently available on the intermediates for the classical induced fit and conformational selection activation pathways. Here these questions are addressed through molecular dynamics simulations on five key states along the thermodynamic cycle for the cAMP-dependent activation of a fully functional construct of EPAC2, which includes the cAMP-binding domain and the integral catalytic region. The simulations are not only validated by the agreement with the experimental trends in cAMP-binding domain dynamics determined by NMR, but they also reveal unanticipated dynamic attributes, rationalizing previously unexplained aspects of EPAC activation and autoinhibition. Specifically, the simulations show that cAMP binding causes an extensive perturbation of dynamics in the distal catalytic region, assisting the recognition of the Rap1b substrate. In addition, analysis of the activation intermediates points to a possible hybrid mechanism of EPAC allostery incorporating elements of both the induced fit and conformational selection models. In this mechanism an entropy compensation strategy results in a low free-energy pathway of activation. Furthermore, the simulations indicate that the autoinhibitory interactions of EPAC are more dynamic than previously anticipated, leading to a revised model of autoinhibition in which dynamics fine tune the stability of the autoinhibited state, optimally sensitizing it to cAMP while avoiding constitutive activation.Journal of Biological Chemistry 08/2011; 286(49):42655-69. · 4.65 Impact Factor
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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.The Journal of General Physiology 06/2011; 137(6):591-603. · 4.73 Impact Factor