Ligand-Induced Conformational Changes in Cyclic Nucleotide Phosphodiesterases and Cyclic Nucleotide-Dependent Protein Kinases

Structural and Functional Features in Human PDE5A1 Regulatory Domain That Provide for Allosteric cGMP Binding, Dimerization, and Regulation

“…By stoichiometric determinations, apparently one cGMP is bound per monomer of PDEs 2, 5, and 6, but the cGMP-binding properties of these PDEs are kinetically heterogeneous (12,20,59,60,(67)(68)(69)(70). Several lines of evidence indicate that PDE5 holoenzyme and its isolated R domain exhibit multiple conformations that relate to alterations in catalytic and/or allosteric cGMP-binding activity (14,27,45,50,51). The resolution of different conformers of PDE5 and isolated R domain on native PAGE, the biphasic kinetics of cGMP dissociation from PDE5 holoenzyme or isolated R domain, and the biphasic dissociation characteristics of inhibitors from PDE5 catalytic site are compelling arguments for conformational/functional heterogeneity.…”

“…Electrophoresis was performed in 25 mM Tris, 192 mM glycine running buffer, pH 8.3, and electrophoresis proceeded for 40 min at constant voltage (250 V) at room temperature according to the manufacturer's instructions. For studies of GAF b, the native gel was prepared as described previously (45) in the presence or absence of 1 mM cGMP in the gel and running buffer, and electrophoresis was conducted at constant voltage (50 V) at 4°C for 24 h. In all instances, bovine serum albumin standard for native gels (Sigma) was run alongside to serve as markers. In experiments utilizing [ 32 P]ATP for phosphorylation of PDE5 constructs, the dye front was run into the lower buffer to remove [ 32 P]ATP prior to staining and autoradiography.…”

“…The molecular events that provide for this cGMP regulation of Ser 102 phosphorylation and the associated increase in allosteric cGMP-binding affinity occur within the context of the R domain (27). Cyclic GMP binding to the allosteric sites also causes an apparent elongation of the enzyme (27,45) and increases the affinity of the PDE5 C domain for substrate and inhibitors even in the absence of phosphorylation (43, 46 -50). Therefore, regulation of the autoinhibition/ activation of PDE5 catalytic function by allosteric cGMP binding and/or phosphorylation involves interactions among multiple subdomains within the R domain.…”

Molecular mechanisms that could contribute to prolonged effectiveness of PDE5 inhibitors to improve erectile function

“…11,[35][36][37] Cyclic GMP-activated PKG undergoes autophosphorylation, which slightly elevates the enzyme's activity even in the absence of cGMP and increases its sensitivity to subsequent increases in cGMP (Figure 3). [38][39][40][41] Autophosphorylation also causes an apparent conformational change in the enzyme and promotes higher affinity for cGMP binding compared to unphospho-PKG, thereby retaining cGMP at the allosteric cGMP-binding sites; 38,42 since the cGMP-binding allosteric sites of PKG are positively cooperative, 43 this would tend to 'prime' PKG for full activation by a subsequent small increase in cGMP.…”

Cyclic GMP Phosphodiesterase-5: Target of Sildenafil

“…All of the components required for catalytic activity of PDE5 are contained within a single monomeric catalytic domain. 6 Occupation of the allosteric cGMP-binding sites of PDE5 is required for specific phosphorylation of Ser-92 by PKG or PKA, and occupation of the binding sites is also associated with an increase in the Stokes radius of the enzyme, implying that a conformational change occurs (53). A direct effect of cGMP binding to the allosteric sites on cGMP breakdown at the catalytic site has not been demonstrated, although the principle of reciprocity (binding of cGMP at the catalytic site stimulates binding at the allosteric sites) dictates that there should be an effect (54,55).…”