The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding

Abstract

Cyclic nucleotide phosphodiesterases (PDEs) regulate all pathways that use cGMP or cAMP as a second messenger. Five of the 11 PDE families have regulatory segments containing GAF domains, 3 of which are known to bind cGMP. In PDE2 binding of cGMP to the GAF domain causes an activation of the catalytic activity by a mechanism that apparently is shared even in the adenylyl cyclase of Anabaena, an organism separated from mouse by 2 billion years of evolution. The 2.9-Å crystal structure of the mouse PDE2A regulatory segment reported in this paper reveals that the GAF A domain functions as a dimerization locus. The GAF B domain shows a deeply buried cGMP displaying a new cGMP-binding motif and is the first atomic structure of a physiological cGMP receptor with bound cGMP. Moreover, this cGMP site is located well away from the region predicted by previous mutagenesis and structural genomic approaches. C yclic nucleotide phosphodiesterases (PDEs) catalyze the hydrolysis of 3Ј, 5Ј cyclic nucleotides to the inactive 5Ј monophosphates. Five of the 11 PDE families contain regulatory segments consisting of one or two so-called GAF-domain modules (1), which is one of the largest families of small moleculebinding regulatory domains. Among PDEs, cGMP is the only ligand known to bind this domain. The structure of a single GAF domain from a putative protein from yeast (YKG9) has been solved recently (2). However, yeast do not make cGMP, nor does this protein bind cGMP when tested directly (2). cGMP binding to one of two GAF domains (3) in the photoreceptor PDE6 family provides one mechanism for regulating visual signal transduction. cGMP also binds to one or more of the GAF domains of PDE5 (4), the target of the drug, Viagra. The binding and subsequent phosphorylation of an adjacent domain activates the catalytic domain of the enzyme (5). In PDE2A, the catalytic activity is allosterically stimulated by cGMP binding to its GAF domain (6), an event important for several pathways that PDE2A has been shown to regulate (7-12). For example, atrial natriuretic peptide stimulation of cGMP and subsequent activation of PDE2A in the adrenal cortex decreases the secretion of aldosterone and, thereby, mediates much of the effect of this hormone on blood pressure (13). Each PDE2A monomer contains an N-terminal (Ϸ200 residues) domain of unknown function, tandem GAF domains (GAF A and GAF B), and a C-terminal catalytic domain. What seems to be a functionally very similar tandem set of GAF domains is also present in Anabaena adenylyl cyclase. This GAF domain has a preference for cAMP where it functions to confer cAMP activation of cyclase activity (14). Here, we report the 2.9-Å crystal structure of the regulatory segment of murine PDE2A, which reveals the structure of two GAF domains with entirely different functions, dimerization, and binding of cGMP. Amazingly, this binding motif and mechanism has apparently been preserved for over 2 billion years in evolution. Methods and MaterialsCrystallization and Data Collection. Crystals were grown at ...

Phosphodiesterases link the aryl hydrocarbon receptor complex to cyclic nucleotide signaling

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“…Five of the 11 PDE families contain regulatory segments consisting of one or two GAF-domain modules (PDE2, 5, 6, 10 and 11). The x-ray crystal structure of murine PDE2A regulatory segment shows that the GAF A domains from each monomer form the dimer interface, while the GAF B domains are well separated and responsible for cGMP binding [73]. PDE2A is highly expressed in brain, heart, platelets, adrenal medulla and endothelial cells, but it is also found in lung, kidney and liver.…”

Characteristics of Photoreceptor PDE (PDE6): similarities and differences to PDE5

“…For both PDE families, the two GAF domains and the catalytic domain are each likely to fold into independent globular structures, as predicted by the crystal structures of the catalytic domain of PDE4, 12 and the tandem GAF domains of PDE2. 13 …”

Interaction of caspase‐3 with the cyclic GMP binding cyclic GMP specific phosphodiesterase (PDE5a1)

“…Indeed, we have shown that in the presence of the inhibitory protein (PDEc) of the rod photoreceptor PDE6, PDE5A1 is a substrate for a low activity preparation of purified caspase-3 [8]. Sitedirected mutagenesis studies have defined the position of the GAF domains [9][10][11] and key amino acid residues involved in the metal ion coordination and catalytic activity [12][13][14][15]. These are shown in Fig.…”