Molecular determinants for cyclic nucleotide binding to the regulatory domains of phosphodiesterase 2A.
Binding of cGMP to the GAF-B domain of phosphodiesterase 2A allosterically activates catalytic activity. We report here a series of mutagenesis studies on the GAF-B domain of PDE2A that support a novel mechanism for molecular recognition of cGMP. Alanine mutations of Phe-438, Asp-439, and Thr-488, amino acids that interact with the pyrimidine ring, decrease cGMP affinity slightly but increase cAMP affinity by up to 8-fold. Each interaction is required to provide for cAMP/cGMP specificity. Mutations of any of the residues that interact with the phosphate-ribose moiety or the imidazole ring abolish cGMP binding. Thus, residues that interact with the pyrimidine ring collectively control cAMP/cGMP specificity, whereas residues that bind the phosphate-ribose moiety and imidazole ring are critical for high affinity binding. Similar decreases in binding were found for mutations made in a bacterially expressed GAF-A/B plus catalytic domain construct. Because these constructs had very high catalytic activity, it appears that these mutations did not cause a global denaturation. The affinities of cAMP and cGMP for wild-type GAF-B alone were approximately 4-fold greater than for the holoenzyme, suggesting that the presence of neighboring domains alters the conformation of GAF-B. More importantly, the PDE2A GAF-B, GAF-A/B, GAF-A/B+C domains, and holoenzyme all bind cGMP with much higher affinity than has previously been reported. This high affinity suggests that cGMP binding to PDE2 GAF-B activates the enzyme rapidly, stoichiometrically, and in an all or none fashion, rather than variably over a large range of cyclic nucleotide concentrations.
Wu
Albert Y
AY
Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA.
Tang
Xiao-Bo
XB
Martinez
Sergio E
SE
Ikeda
Kaori
K
Beavo
Joseph A
JA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002384:Catalysis; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; D017931:DNA Primers; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed
D017931:DNA Primers; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; C517277:Pde2a protein, mouse; D006152:Cyclic GMP