Solution Structure of the cGMP Binding GAF Domain from Phosphodiesterase 5

Abstract

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 ␤-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 ϳ20 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.Intracellular concentrations of the second messengers cAMP and cGMP are tightly regulated by the rate of synthesis through cyclases and hydrolysis through cyclic nucleotide phosphodiesterases (PDEs) 3 (1). The cGMP-specific, cGMP-binding phosphodiesterase, PDE5, is one of eleven identified PDE families. It has been characterized as the major cGMP-hydrolyzing PDE in numerous tissues such as lung, platelets, pulmonary artery smooth muscle cells, and the penile corpus cavernosum (2, 3). The abundance of PDE5 in smooth muscles and its role in regulating their contractile tone has made PDE5 an important drug target for the treatment of erectile dysfunction and pulmonary hypertension (2), leading to the development of potent PDE5 inhibitors, such as tadalafil (Cialis TM

Distinct Allostery Induced in the Cyclic GMP-binding, Cyclic GMP-specific Phosphodiesterase (PDE5) by Cyclic GMP, Sildenafil, and Metal Ions

“…1D), similar to that reported for native PDE5 (12). Addition of cGMP to lysates prepared from cells in the presence of EDTA led to a decrease (ϳ40%) in BRET of the wildtype sensor, whereas mutation of the Phe-163 residue (which has a stacking interaction with cGMP in the GAFa domain (27,35)) to alanine abrogated the BRET decrease ( Fig. 2A).…”

“…Sodium ions have been shown to regulate cNMP binding to the GAF domains of CyaB2 and PDE2 (43). Analysis of the available structures of the GAFa and the catalytic domain of PDE5 (35,38) showed potential disulfide pairing of cysteine residues, suggesting the possibility of redox regulation of PDE5 conformation in cells. Additionally, PDE5 requires two divalent cations (Mg 2ϩ and Zn 2ϩ ) bound to the catalytic domain for cGMP hydrolysis, and these metal ions could control the conformation of PDE5 (38).…”

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

“…In contrast, the shorter sequence between strands ␤2 and ␤3 in PDE2A GAF B does not allow for formation of the ␣2/3 helix, and there are no analogous contacts with Asp-439, thereby allowing the residue to swing out and cAMP to bind more easily. PDE5A with its thousandfold cGMP preference also contains a ␣2/3 helix in similar orientation (35).…”

“…In particular, the side chains of Ser-121 and Asn-126 make hydrogen bonds to the backbone carbonyl of Asn-116 and may help to lock its side and main chains into position to provide the nucleotide-selective hydrogen bonds. A solution structure of PDE5A GAF A revealed that the cGMP-selective GAF domain of PDE5A contains a similar helical turn (35).…”

“…Recently, our laboratories investigated the cGMP-induced conformational change of GAF A from PDE5A (35). In contrast to apo-GAF A of PDE6C, apo-GAF A of PDE5A is not stable in solution.…”