Structure and regulation of the cAMP-binding domains of Epac2

Abstract

Cyclic adenosine monophosphate (cAMP) is a universal second messenger that, in eukaryotes, was believed to act only on cAMP-dependent protein kinase A (PKA) and cyclic nucleotide-regulated ion channels. Recently, guanine nucleotide exchange factors specific for the small GTP-binding proteins Rap1 and Rap2 (Epacs) were described, which are also activated directly by cAMP. Here, we have determined the three-dimensional structure of the regulatory domain of Epac2, which consists of two cyclic nucleotide monophosphate (cNMP)-binding domains and one DEP (Dishevelled, Egl, Pleckstrin) domain. This is the first structure of a cNMP-binding domain in the absence of ligand, and comparison with previous structures, sequence alignment and biochemical experiments allow us to delineate a mechanism for cyclic nucleotide-mediated conformational change and activation that is most likely conserved for all cNMP-regulated proteins. We identify a hinge region that couples cAMP binding to a conformational change of the C-terminal regions. Mutations in the hinge of Epac can uncouple cAMP binding from its exchange activity.

Structural snapshot of cyclic nucleotide binding domains from cyclic nucleotide-sensitive ion channels

“…The β roll is surrounded by the N-terminal α-helix A, a short internal α-helix located between β-strands 6 and 7, designated as the phosphate-binding-cassette (PBC), and two α-helices B and C (αB and αC) located at the C-terminus. This structure, together with previously determined structures of PKA, CAP and Epac, confirmed the β roll topology to be conserved in all CNBDs (Weber and Steitz, 1987;Su et al, 1995;Diller et al, 2001;Rehmann et al, 2003).…”

“…The structure of the MloK1 CNBD shows the characteristic properties similar to the HCN2 CNBD structure (Figures 1 and 2), consisting of a β roll core topped by a bundle of four α-helices and a short PBC helix (Zagotta et al, 2003). Thus, the overall structure is highly conserved in all CNBDs (Weber and Steitz, 1987;Su et al, 1995;Diller et al, 2001;Rehmann et al, 2003).…”

“…Interestingly, an alternative CNBD dimerization interface was reported (Gushchin et al, 2012), which is present in the MloK1 CNBD point mutant R307W homodimer Figure 5C) and the Epac2 heterodimer (Rehmann et al, 2003(Rehmann et al, , 2006 crystal structures. In contrast to the reported predominant MloK1 CNBD dimer interface present in the crystal structure (Clayton et al, 2004), molecular dynamics simulations showed that the novel interface is stable and no structural rearrangements were observed.…”

“…Cyclic nucleotides bind to the cyclic nucleotide-binding domain (CNBD) located at the C-terminus of each subunit. These CNBDs have a conserved fold when compared with other cyclic nucleotide-binding proteins, such as protein kinases G and A (PKG and PKA; Vaandrager and de Jonge, 1996;Kim et al, 2005), the bacterial cAMP-regulated catabolite gene activator protein (CAP) (Zubay et al, 1970;Eron et al, 1971;McKay and Steitz, 1981), the exchange protein directly activated by cAMP (EPAC) (Rehmann et al, 2003), and KCNH channels (Brelidze et al, 2012;Marques-Carvalho et al, 2012).…”

cAMP activation of PKA defines an ancient signaling mechanism

“…1 c and d) propagate through a long-range allosteric network that spans both ␣-and ␤-subdomains? Previous analyses (8-16) have led to the proposal of an initial allosteric model in which the ␣-and ␤-subdomains are directly coupled to each other through a salt bridge between E200 and R241 and also possibly through a hydrophobic hinge defined by the L203, I204, and Y229 side-chain cluster (9,11,12). However, mutations (17), sequence conservation analyses (1), structurebased comparisons (1), and genetic screening (18,19) indicate that several other sites, which are not accounted for by the existing model, are also likely to play an active role in the cAMP-mediated activation of PKA.…”