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.
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The mutations utilized in this study suggest that the previously proposed hydrophobic hinge hypothesis based on the L273/F300 steric clash is not sufficient alone to account for all the molecular determinants of EPAC autoinhibition. First, several other highly conserved motifs within the CBD contribute to autoinhibition, including the β 2- β 3 loop and the α 5 helix. Second, these conserved structural elements fine-tune the autoinhibitory equilibrium of EPAC for optimal cAMP sensitivity not only through steric and van der Waals interactions, as previously proposed, but also through entropic losses arising from regions that are more dynamic in the apo/inactive state relative to the apo/active state. Third, the multiple autoinhibitory determinants identified here appear to be nondegenerate with respect to the complete activation of EPAC, suggesting that their collective suppression is required for full EPAC activity. Such nondegeneracy provides an effective means to enhance the specificity of the allosteric effectors that act on the EPAC molecular switch. Fourth, we anticipate that the concepts and methods illustrated here will be useful also for other systems. For instance, the presumed novel projection analysis (PA) proposed here will likely be useful in evaluating how mutations modulate the position of conformational equilibria for domains that function as allosteric sensors in the regulation of signaling pathways ( 28–30 ). We thank M. Akimoto, J. Milojevic, and Dr. Holger Rehmann for helpful discussions. We also acknowledge the Shared Hierarchical Academic Research Computing Network for use of their high-performance computing resources as well as Dr. Stuart Rothstein's research group for use of the Procrustean rotation software. We thank the Canadian Institute of Health Research and the National Sciences and Engineering Research Council for financial support. We are also indebted to the Heart and Stroke Foundation of Canada for a Maureen Andrew New Investigator award to G.M.