Structural analyses of a constitutively active mutant of exchange protein directly activated by cAMP.

Rafael Josef Najmanovich,Mark A White,Tamara Tsalkova,Virgil L Woods,Fang C Mei,Xiaodong Cheng,Sheng Li,Tong Liu

doi:10.1371/journal.pone.0049932

Abstract

Exchange proteins directly activated by cAMP (EPACs) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we have combined site-directed mutagenesis, X-ray crystallography, and peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) to probe the structural and conformational dynamics of EPAC2-F435G, a constitutively active EPAC2 mutant. Our study demonstrates that conformational dynamics plays a critical role in cAMP-induced EPAC activation. A glycine mutation at 435 position shifts the equilibrium of conformational dynamics towards the extended active conformation.

Highlights

Exchange proteins directly activated by cAMP (EPACs) are an important family of signaling molecules serving as the intracellular sensors for the prototypic second messenger [1,2]
Even though EPAC2-F435G is active in solution without cAMP, the apo-EPAC2-F435G crystal structure still represents the compact, inactive apo form of EPAC2, trapped by the crystal lattice, which is incompatible with the extended, active conformation
While structural changes immediately adjacent to the site of mutation between WT and EPAC2-F435G are relatively small, major structural deviations occur at distal sites, at the C-terminal catalytic lobe, suggesting global allosteric effects of the mutation (Figure 1)

Summary

Introduction

Exchange proteins directly activated by cAMP (EPACs) are an important family of signaling molecules serving as the intracellular sensors for the prototypic second messenger [1,2]. The two mammalian EPAC isoforms, EPAC1 and EPAC2, share extensive sequence and structural homology, which includes a conserved Cterminal catalytic core that consists of a RAS exchange (REM) domain, a RAS association (RA) domain, and a CDC25-homology guanine nucleotide exchange factor (GEF) domain. While both the N-terminal regulatory region of EPAC1 and EPAC2 contain a Dishevelled-Egl-Pleckstrin (DEP) domain and a cAMP binding domain (CBD), EPAC2 has an additional CBD in front of the DEP domain (Figure 1A). When the intracellular concentration of cAMP rises, it binds to the cAMP binding domain (CBD) of EPAC and induces conformational changes, in the hinge and switchboard (SB), that lead to activation of EPAC by exposing the C-terminal catalytic core, which interacts with and activates down-stream effectors, Rap or Rap2 [1,2] partially through residues in the Helical Hairpin (HP) [4]

Methods

Results

Conclusion