Structural Features of the Noncatalytic cGMP Binding Sites of Frog Photoreceptor Phosphodiesterase Using cGMP Analogs

Abstract

The cGMP-specific phosphodiesterase (PDE) of retinal photoreceptors is a central regulatory enzyme in the visual transduction pathway of vertebrate vision. Although the mechanism of activation of PDE by transducin is well understood, the role of the noncatalytic cGMP binding sites located on the catalytic subunits of PDE remains obscure. We report here for the first time the molecular basis of the noncovalent interactions between cGMP and the high affinity, noncatalytic cGMP binding sites of frog photoreceptor PDE.None of the tested cGMP analogs were able to bind with greater affinity than cGMP itself, and the noncatalytic sites were unable to bind cAMP. The major determinant for discrimination of cGMP over cAMP is in the N-1/C-6 region of the purine ring of cGMP where hydrogen bonding probably stabilizes the selective binding of cGMP. Substitutions at the C-2 position demonstrate that this region of the molecule plays a secondary but significant role in stabilizing cGMP binding to PDE through hydrogen bond interactions. The unaltered hydrogen at the C-8 position is also important for high affinity binding. A significant interaction between the binding pocket and the ribose ring of cGMP occurs at the 2-hydroxyl position. Steric constraints were greatest in the C-8 and possibly the C-6/N-1 regions, whereas the C-2/N-3 and C-2 regions tolerated bulky substituents better. Several lines of evidence indicate that the noncatalytic site binds cGMP in the anti-conformation. The numerous noncovalent interactions between cGMP and the noncatalytic binding pocket of the photoreceptor PDE described in this study account for both the high affinity for cGMP and the high level of discrimination of cGMP from other cyclic nucleotides at the noncatalytic site.Visual excitation of vertebrate retinal photoreceptors begins with the absorption of light by the visual pigment (opsin) which is followed by activation of a G-protein, transducin. This results in activation of a photoreceptor-specific cGMP phosphodiesterase (PDE).1 Increased cGMP hydrolysis lowers the cytoplasmic cGMP concentration which is believed to cause closure of the cGMP-gated ion channel in the plasma membrane and the generation of an electrical response (for reviews, see Refs. 1-5).The rod photoreceptor PDE holoenzyme is a heterotetramer consisting of two non-identical catalytic subunits (␣ and ␤) and two small subunits (␥) that serve to inhibit catalytic activity. Cone photoreceptors have a highly homologous PDE that consists of two identical catalytic subunits (␣Ј) and two conespecific ␥Ј subunits. In addition to containing the active site for cGMP hydrolysis, the catalytic subunits of rod and cone PDEs also possess noncatalytic cGMP binding sites (6). Two other classes of PDEs, namely the cGMP-stimulated PDE (PDE2) and the cGMP-specific PDE (PDE5), are also known to contain noncatalytic binding sites for cGMP. In the case of PDE2, binding of cGMP at the noncatalytic site allosterically activates cyclic nucleotide hydrolysis at the active site (7,8). For PDE5, the...

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

“…The general features of cGMP binding in the GAF B structure are consistent with many of the predictions made from binding studies of PDE2 and PDE6 with cyclic nucleotide analogues. These studies suggested that cGMP would be bound in the anti-conformation, that contacts are made to C6, N1, and C2 amino group of the guanine ring and 2ЈO of the ribose ring, and that there are similar or identical types of contacts to the exocyclic oxygens (21,26,34). (D) cGMP and cAMP competition binding curves [against ( 3 H)cGMP] for the wild-type regulatory segment of mouse PDE2A.…”

Properties and Functions of GAF Domains in Cyclic Nucleotide Phosphodiesterases and Other Proteins

“…In PDE2, the cGMP-versuscAMP selectivity of the allosteric cN-binding sites is considerably lower, and other molecules may also interact with these sites to stimulate catalytic activity (Erneux et al, 1985;Manganiello et al, 1990;Martinez et al, 2002b). The specificity requirements for cGMP binding to the GAF(s) in PDEs 5 and 6 are the most restrictive of any known cN-binding protein, and these sites tolerate very few cN analogs (Francis et al, 1990;Hebert et al, 1998).…”

“…In PDE2, cGMP is bound in the anticonformation of the N-glycosidic linkage, and the cyclic phosphate moiety is in the energetically unfavorable boat conformation. Earlier studies using cGMP analogs with PDE5 and PDE6 had predicted that cGMP is bound in the anti-conformation (Francis et al, 1990;Hebert et al, 1998).…”

“…In PDEs 5 and 6, the importance of the 2Ј-OH interaction in allosteric cGMP binding has been demonstrated by analog studies (Francis et al, 1990;Hebert et al, 1998), but the crystal structure of PDE2 GAF b domain does not reveal a bond with this position. In addition, the allosteric cN-binding sites in PDE5 and PDE6 have much higher specificity for cGMP versus cAMP compared with PDE2, and PDE6 binds cGMP with higher affinity than does the PDE2 or PDE5 (Erneux et al, 1985;Beavo, 1988, 1989;Thomas et al, 1990a;Cote et al, 1994).…”

“…GAF domains in PDEs 5 and 6 have more stringent specificity toward cGMP and are less likely to interact with cAMP under physiological conditions. Cyclic GMP binds to the photoreceptor PDE family (PDE6A-C) with higher intrinsic affinity than to either PDE2 or PDE5 (Erneux et al, 1985;Beavo, 1988, 1989;Thomas et al, 1990a;Cote et al, 1994;Hebert et al, 1998). Comparison of the cGMP-binding contacts derived from the PDE2 GAF b with sequences in the GAFs of PDEs 5 and 6 led Beavo and colleagues to predict that, like PDE5, cGMP-binding in PDE6 most likely occurs in GAF a (Martinez et al, 2002b).…”