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Originally published In Press as doi:10.1074/jbc.M413611200 on January 27, 2005

J. Biol. Chem., Vol. 280, Issue 12, 12051-12063, March 25, 2005
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Structural and Functional Features in Human PDE5A1 Regulatory Domain That Provide for Allosteric cGMP Binding, Dimerization, and Regulation*

Roya Zoraghi, Emmanuel P. Bessay, Jackie D. Corbin, and Sharron H. Francis{ddagger}

From the Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615

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 (KD < 30 nM) and between GAF b modules (KD = 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 (KD < 40 nM) than does the isolated R domain (KD = 110 nM) or holoenzyme (KD = 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.

Received for publication, December 3, 2004 , and in revised form, January 25, 2005.

* This work supported by National Institutes of Health Research Grants DK40299 and DK58277 and by American Heart Association Postdoctoral Fellowship 032525B. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{ddagger} To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 702 Light Hall, Nashville, TN 37232-0615. Tel.: 615-322-4383; Fax: 615-343-3794; E-mail: Sharron.Francis{at}vanderbilt.edu.


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