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.
Highlights
EXPERIMENTAL PROCEDURESMaterials—Frogs were obtained from Charles Sullivan, Inc. Multiscreen filtration plates (MAHA N45) were purchased from Millipore
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 principal finding of this paper is that the noncatalytic cGMP binding pocket on the photoreceptor PDE interacts in a highly specific manner with the purine ring, the ribose ring, and with the cyclic phosphate ring of cGMP (Fig. 3)
Summary
Materials—Frogs were obtained from Charles Sullivan, Inc. Multiscreen filtration plates (MAHA N45) were purchased from Millipore. Preparation of N2,3-Etheno-cGMP (10)—Compound 6 (80 mol) was added to 10 ml of 70% methanol (in water) in the presence of 10% palladium on charcoal (50 mg); the sample was completely hydrogenated after 40 min at room temperature, as observed by analytical HPLC. Portions of homogenized ROS (4 M rhodopsin, 12 nM PDE holoenzyme) were mixed with 60 nM [3H]cGMP (6.64 ϫ 1016 dpm/mol; prepared in Buffer A) which had been premixed with the indicated concentration of a cyclic nucleotide analog. This concentration of [3H]cGMP was chosen such that ϳ50% of the high affinity noncatalytic sites on PDE would be occupied in the absence of competing nucleotides. For all pairwise comparisons of cGMP analogs discussed in the text, the Ki values were determined to be statistically significant at the p Ͻ 0.005 level of significance using a paired t test
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