Abstract
Exchange proteins directly activated by cAMP (EPAC1 and EPAC2) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we performed detailed Small-Angle X-ray Scattering (SAXS) analysis of EPAC1 in its apo (inactive), cAMP-bound, and effector (Rap1b)-bound states. Our study demonstrates that we can model the solution structures of EPAC1 in each state using ensemble analysis and homology models derived from the crystal structures of EPAC2. The N-terminal domain of EPAC1, which is not conserved between EPAC1 and EPAC2, appears folded and interacts specifically with another component of EPAC1 in each state. The apo-EPAC1 state is a dynamic mixture of a compact (Rg = 32.9 Å, 86%) and a more extended (Rg = 38.5 Å, 13%) conformation. The cAMP-bound form of EPAC1 in the absence of Rap1 forms a dimer in solution; but its molecular structure is still compatible with the active EPAC1 conformation of the ternary complex model with cAMP and Rap1. Herein, we show that SAXS can elucidate the conformational states of EPAC1 activation as it proceeds from the compact, inactive apo conformation through a previously unknown intermediate-state, to the extended cAMP-bound form, and then binds to its effector (Rap1b) in a ternary complex.
Highlights
In vertebrates, the majority of the intracellular functions of the pleiotropic second messenger cAMP are mediated by the exchange proteins directly activated by cAMP (EPACs) and cAMP-dependent protein kinase (PKA)
Mammalian EPACs are found as two major isoforms, EPAC1 and EPAC2, which share significant sequence and structural homology, differing mostly via an N-terminal truncation of the first cAMP
In the absence of Rap1, the cAMP-bound EPAC1 proteins dimerize under the conditions required for solution scattering studies
Summary
The majority of the intracellular functions of the pleiotropic second messenger cAMP are mediated by the exchange proteins directly activated by cAMP (EPACs) and cAMP-dependent protein kinase (PKA). EPAC and PKA share a homologous cyclic nucleotide binding domain (CNBD) [1]. Mammalian EPACs are found as two major isoforms, EPAC1 and EPAC2, which share significant sequence and structural homology, differing mostly via an N-terminal truncation of the first cAMP domain (CNBD-A) in EPAC1 (Figure 1) [2,3]. While purified recombinant EPAC1 and 2 proteins have similar biochemical activities in vitro, in terms of their abilities to response to cAMP stimulation and to activate down-stream effectors Rap and Rap, their physiological functions are largely non-redundant. The N-terminal sequence variation between EPAC1 and EPAC2 plays an important role in their functional diversities. The CNBD-A of EPAC2, while very poor at binding cAMP, is critical for directing EPAC2 to the granule sites in β-cells [5]
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