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J. Biol. Chem., Vol. 283, Issue 33, 22749-22759, August 15, 2008

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Solution Structure of the cGMP Binding GAF Domain from Phosphodiesterase 5

INSIGHTS INTO NUCLEOTIDE SPECIFICITY, DIMERIZATION, AND cGMP-DEPENDENT CONFORMATIONAL CHANGE^*

Clemens C. Heikaus, Joseph R. Stout, Monica R. Sekharan¹, Catherine M. Eakin, Ponni Rajagopal, Peter S. Brzovic, Joseph A. Beavo, and Rachel E. Klevit²

From the Departments of Biochemistry and Pharmacology, University of Washington, Seattle, Washington 98195

Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular nuclear Overhauser effects. Comparison with GAF domains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall domain fold of a six-stranded β-sheet and four -helices that form a well defined cGMP binding pocket. However, the nucleotide coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound structural conformation. NMR studies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and indicate a large conformational change upon ligand binding. Furthermore, we identify a region of 20 residues directly N-terminal of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific regulators of PDE5 function.

Received for publication, February 27, 2008 , and in revised form, April 17, 2008.

The atomic coordinates and structure factors (code 2k31) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported, in whole or in part, by National Institutes of Health Grant 1 P01 HL44948 (to R. E. K. and J. A. B.). This work was also supported by a Boehringer Ingelheim Fonds Ph.D. Scholarship (to C. C. H.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental text, references, Figs. S1-S7, and Table S1.

¹ Dept. of Chemistry and Chemical Biology, Rutgers University, 610 Taylor Road, Piscataway, NJ 08854.

² To whom correspondence should be addressed: Dept. of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7350. Tel.: 206-543-5891; Fax: 206-543-8394; E-mail: klevit{at}u.washington.edu.

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				S. E. Martinez, C. C. Heikaus, R. E. Klevit, and J. A. Beavo The Structure of the GAF A Domain from Phosphodiesterase 6C Reveals Determinants of cGMP Binding, a Conserved Binding Surface, and a Large cGMP-dependent Conformational Change J. Biol. Chem., September 19, 2008; 283(38): 25913 - 25919. [Abstract] [Full Text] [PDF]