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.
Article: Substrate-Specific Reorganization of the Conformational Ensemble of CSK Implicates Novel Modes of Kinase Function.[show abstract] [hide abstract]
ABSTRACT: Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell.PLoS Computational Biology 09/2012; 8(9):e1002695. · 5.22 Impact Factor
Article: The projection analysis of NMR chemical shifts reveals extended EPAC autoinhibition determinants.[show abstract] [hide abstract]
ABSTRACT: EPAC is a cAMP-dependent guanine nucleotide exchange factor that serves as a prototypical molecular switch for the regulation of essential cellular processes. Although EPAC activation by cAMP has been extensively investigated, the mechanism of EPAC autoinhibition is still not fully understood. The steric clash between the side chains of two conserved residues, L273 and F300 in EPAC1, has been previously shown to oppose the inactive-to-active conformational transition in the absence of cAMP. However, it has also been hypothesized that autoinhibition is assisted by entropic losses caused by quenching of dynamics that occurs if the inactive-to-active transition takes place in the absence of cAMP. Here, we test this hypothesis through the comparative NMR analysis of several EPAC1 mutants that target different allosteric sites of the cAMP-binding domain (CBD). Using what to our knowledge is a novel projection analysis of NMR chemical shifts to probe the effect of the mutations on the autoinhibition equilibrium of the CBD, we find that whenever the apo/active state is stabilized relative to the apo/inactive state, dynamics are consistently quenched in a conserved loop (β2-β3) and helix (α5) of the CBD. Overall, our results point to the presence of conserved and nondegenerate determinants of CBD autoinhibition that extends beyond the originally proposed L273/F300 residue pair, suggesting that complete activation necessitates the simultaneous suppression of multiple autoinhibitory mechanisms, which in turn confers added specificity for the cAMP allosteric effector.Biophysical Journal 02/2012; 102(3):630-9. · 3.65 Impact Factor
[show abstract] [hide abstract]
ABSTRACT: The cyclic-AMP binding domain (CBD) is the central regulatory unit of exchange proteins activated by cAMP (EPAC). The CBD maintains EPAC in a state of auto-inhibition in the absence of the allosteric effector, cAMP. When cAMP binds to the CBD such auto-inhibition is released, leading to EPAC activation. It has been shown that a key feature of such cAMP-dependent activation process is the partial destabilization of a structurally conserved hinge helix at the C-terminus of the CBD. However, the role of this helix in auto-inhibition is currently not fully understood. Here we utilize a series of progressive deletion mutants that mimic the hinge helix destabilization caused by cAMP to show that such helix is also a pivotal auto-inhibitory element of apo-EPAC. The effect of the deletion mutations on the auto-inhibitory apo/inactive vs. apo/active equilibrium was evaluated using recently developed NMR chemical shift projection and covariance analysis methods. Our results show that, even in the absence of cAMP, the C-terminal region of the hinge helix is tightly coupled to other conserved allosteric structural elements of the CBD and perturbations that destabilize the hinge helix shift the auto-inhibitory equilibrium toward the apo/active conformations. These findings explain the apparently counterintuitive observation that cAMP binds more tightly to shorter than longer EPAC constructs. These results are relevant for CBDs in general and rationalize why substrates sensitize CBD-containing systems to cAMP. Furthermore, the NMR analyses presented here are expected to be generally useful to quantitatively evaluate how mutations affect conformational equilibria.PLoS ONE 01/2012; 7(11):e48707. · 4.09 Impact Factor