The auto-inhibitory role of the EPAC hinge helix as mapped by NMR

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.