Abstract
The cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains a catalytic domain that hydrolyzes cGMP and a regulatory (R) domain that contains two GAFs (a and b; GAF is derived from the proteins mammalian cGMP-binding PDEs, Anabaena adenylyl cyclases, and Escherichia coli (FhlA)). The R domain binds cGMP allosterically, provides for dimerization, and is phosphorylated at a site regulated by allosteric cGMP binding. Quaternary structures and cGMP-binding properties of 10 human PDE5A1 constructs containing one or both GAFs were characterized. Results reveal that: 1) high affinity homo-dimerization occurs between GAF a modules (K(D) < 30 nM) and between GAF b modules (K(D) = 1-20 pM), and the sequence between the GAFs (Thr322-Asp403) contributes to dimer stability; 2) 176 amino acids (Val156-Gln331) in GAF a are adequate for cGMP binding; 3) GAF a has higher affinity for cGMP (K(D) < 40 nM) than does the isolated R domain (K(D) = 110 nM) or holoenzyme (K(D) = 200 nM), suggesting that the sequence containing GAF b and its flanking amino acids autoinhibits GAF a cGMP-binding affinity in intact R domain; 4) a mutant (Met1-Glu321) containing only GAF a has high affinity, biphasic cGMP-binding kinetics consistent with structural heterogeneity of GAF a, suggesting that the presence of GAF b is not required for biphasic cGMP-dissociation kinetics observed in holoenzyme or isolated R domain; 5) significant cGMP binding by GAF b was not detected; and 6) the sequence containing GAF b and its flanking amino acids is critical for cGMP stimulation of Ser102 phosphorylation by cyclic nucleotide-dependent protein kinases. Results yield new insights into PDE5 functions, further define boundaries that provide for allosteric cGMP binding, and identify regions that contribute to dimerization.
Highlights
Intracellular levels of cyclic nucleotides1 are largely determined by the balance between the rates of synthesis by
Much evidence supports the interpretation that the effects of allosteric cGMP binding and/or phosphorylation to stimulate PDE5 catalytic activity is critically involved in negative feedback regulation of cellular cGMP levels; the same mechanisms participate in a feed-forward feedback process to increase potency of PDE5 inhibitors [43, 50, 51]
The constructs included the recombinant human PDE5A1 holoenzyme
Summary
Intracellular levels of cyclic nucleotides (cNs) are largely determined by the balance between the rates of synthesis by. Cyclic GMP binding to the allosteric sites 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). 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. Much evidence supports the interpretation that the effects of allosteric cGMP binding and/or phosphorylation to stimulate PDE5 catalytic activity is critically involved in negative feedback regulation of cellular cGMP levels; the same mechanisms participate in a feed-forward feedback process to increase potency of PDE5 inhibitors [43, 50, 51]. Mechanisms by which cGMP binding and phosphorylation of the R domain affect enzyme activity and the roles of individual GAFs in PDE5 are not fully understood
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