Role of Dynamics in the Autoinhibition and Activation of the Exchange Protein Directly Activated by Cyclic AMP (EPAC)

Bryan Vanschouwen,Rajeevan Selvaratnam,Giuseppe Melacini,Federico Fogolari

doi:10.1074/jbc.m111.277723

Bryan Vanschouwen, Rajeevan Selvaratnam + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m111.277723

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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.

Highlights

The structures of exchange protein directly activated by cAMP (EPAC) solved in the absence and presence of a nonhydrolysable agonist closely related to cAMP have shown that EPAC adopts two main conformations, which differ with respect to the relative regulatory region (RR)/catalytic region (CR) orientation (Fig. 1b) [14, 15]
For the purpose of gaining insight into the dynamics of fulllength EPAC in all four states of the thermodynamic cycle of EPAC activation (Fig. 1b), we present here molecular dynamics (MD) simulations (34 –38) on a functional EPAC2 construct that includes both the regulatory cyclic nucleotide-binding (CNB) and the integral catalytic region
The Closed Topology of the Apo/Inactive State and the Fold of Each Individual Domain Are Stable in the Nanosecond Time Scale—As a first assessment of the simulated MD trajectory starting from the apo/inactive structure, the r.m.s. deviations from both active and inactive initial EPAC2 conformations were computed over the course of the simulation (Fig. 2a)

Summary

Introduction

The structures of EPAC solved in the absence and presence of a nonhydrolysable agonist closely related to cAMP have shown that EPAC adopts two main conformations, which differ with respect to the relative RR/CR orientation (Fig. 1b) [14, 15]. The second major change induced by cAMP binding is a breaking of the ionic latch interactions between the NTHB of the CNB domain and the catalytic site of the CDC25-HD, assisting the displacement of the EPAC regulatory region away from the CDC25-HD via the hinge rotation [14, 15]. Without a full map of the dynamic profile of EPAC, key aspects of EPAC function remain unexplained Another limitation of the current experimental investigations on EPAC is that they are to a large extent limited to the apo/inactive and holo/active states due to the effective coupling between cAMP binding and the closed-to-open conformational transition. No information is currently available on the apo/active and holo/inactive cross-states (“metastates”), which represent key intermediates in the thermodynamic cycle that models the coupling between cAMP binding and allosteric conformational changes (Fig. 1b). The characterization of the metastates is essential to dissect the distinct contributions of cAMP binding and conformational changes to the variations in dynamics between the apo-inactive and holo-active states

Results

Discussion

Conclusion