Replacement of the L273 residue on the PBC with a bulky tryptophan mutation prevents the hinge adopting the active conformation, and no GEF activity was observed in EPAC1 L273W , even in the presence of 500 µM cAMP. The interface between the RR and the CR is also stabilized via a mixture of hydrogen bonding and ionic interactions between residues of the two domains (Supplementary Figure ) (ionic latch, or IL), and the helical conformation of the hinge prevents opening of EPAC1. Upon binding of cAMP, the PBC shifts from an "out" to an "in" conformation (Figure ). This allows the hinge helix to unfold at its C-terminus end, moving the RR by approximately 45 Å and enabling RAP binding to the exposed active site (Figure ). Additionally, the active conformation of EPAC1 is further stabilized by the interactions between the adenosine group of cAMP and K353 of the REM domain on the catalytic region, as well as E315 of the C-terminus of the cNBD (Figure ). The terminal β-sheet strands of the cNBD and the first helix of the REM domain are known as the "lid", as they close off the previously solvent-exposed cAMP binding site upon activation. To summarise, the two main structural features that discriminate between EPAC1 inactive and active conformations (Figure ) are: the PBC region ("out" to "in" transition), and the hinge region ("out" to "in" transition).