Modeling and mutational analysis of the GAF domain of the cGMP-binding, cGMP-speci¢c phosphodiesterase, PDE5

Abstract The GAFa domain of the cGMP-binding, cGMP-speci¢c phosphodiesterase (PDE5A) was modeled on the crystal structure of PDE2A GAF domain and residues involved in cGMP binding identi¢ed. Tandem GAFa and GAFb domains of PDE5A, expressed in Escherichia coli, bound cGMP (Kd 27 nM). Mutation of aspartate-299 in GAFa, suggested earlier to be critical for cGMP binding, did not abrogate cGMP binding, but mutation of F205, which formed a stacking interaction with the guanine ring of cGMP, led to complete loss of cGMP bind-ing. Therefore, the GAFa domain of PDE5A adopts a structure similar to the GAFb domain of PDE2A, and provides the sole site for cGMP binding in PDE5A