Conformation changes, N-terminal involvement, and cGMP signal relay in the phosphodiesterase-5 GAF domain

Abstract

The activity of phosphodiesterase-5 (PDE5) is specific for cGMP and is regulated by cGMP binding to GAF-A in its regulatory domain. To better understand the regulatory mechanism, x-ray crystallographic and biochemical studies were performed on constructs of human PDE5A1 containing the N-terminal phosphorylation segment, GAF-A, and GAF-B. Superposition of this unliganded GAF-A with the previously reported NMR structure of cGMP-bound PDE5 revealed dramatic conformational differences and suggested that helix H4 and strand B3 probably serve as two lids to gate the cGMP-binding pocket in GAF-A. The structure also identified an interfacial region among GAF-A, GAF-B, and the N-terminal loop, which may serve as a relay of the cGMP signal from GAF-A to GAF-B. N-terminal loop 98-147 was physically associated with GAF-B domains of the dimer. Biochemical analyses showed an inhibitory effect of this loop on cGMP binding and its involvement in the cGMP-induced conformation changes.

FIGURE 1.

Ribbon diagram of the GAF domains of PDEs. A, parallel dimer of the GAF domain of human PDE5A1 fragment 98–518. The blue spheres below the label H5 represent Cys³¹⁰, where the helix bends. Small pink spheres below the label B1 represent Ser¹⁹⁴ and Asn⁴⁸⁵. cGMP marks the cGMP-binding pocket. Helix H0 (blue) is an artifact resulting from the expression vector. B, superposition of the PDE5A dimer (green sticks) with unliganded PDE2A (yellow; Protein Data Bank code 3IBJ) (28) by secondary structure matching. Although the central helices of the two dimers are well superimposed, many portions of the structures are significantly off their positions. C, superposition of the PDE5A monomer (gold) with the cGMP-bound GAF domain of mouse PDE2A (green; Protein Data Bank code 1MCO) (14). Two GAF-B domains in the cGMP-bound PDE2A dimer switched their locations.

FIGURE 2.

Conformational changes upon cGMP binding. A, superposition of GAF-A of the x-ray PDE5A1 structure (green) with that of NMR (gold and cyan). The gold and cyan colors represent large and small positional differences, respectively. The purple sticks represent cGMP. B, model of cGMP binding to the “open” pocket of GAF-A of unliganded PDE5A. C, upon cGMP binding, helix H4 and strand B3 move toward one another and thus serve as lids to gate the binding pocket.

FIGURE 3.

Interaction of N-terminal loop 101–127 of PDE5A1 with GAF-A and GAF-B. Left, surface plot of the interactions. Subunits A and B of the dimer are plotted on the left and right. The pink helix is composed of 8 residues from the expression vector and is an artifact. Residues 98–106 of subunit A (pink helix and spheres) are located in the front of GAF-B, but residues 107–127 turn to the back of GAF-B. Residues 115–124 form two short β-strands in subunit B (purple arrows), but coils in GAF-A. Ser¹⁰² (spheres) is a phosphorylation residue and spatially accessible, but is hindered by the proximity to GAF-A. Center, stereoview of interactions among the N-terminal loop (cyan), GAF-A (green sticks and ribbons), and GAF-B (gold) within the same subunit. The dashed lines represent hydrogen bonds. Right, interactions of the N-terminal loop (light cyan) with GAF-B domains. (Left) and (Center) denote segments from subunits A and B. Ser¹²³–Asp¹²⁷ of subunit B interact mainly with residues from subunit A (yellow) via hydrogen bonds and hydrophobic interactions. In addition, Phe¹²⁴ contacts Phe⁴⁹⁸ within the same subunit.

FIGURE 4.

Native acrylamide gels of PDE5A1(C149S) fragments. A, native gel of PDE5A1(C149S) (amino acids 89–518). Samples were treated with various concentrations of cGMP or with 2 mm cAMP and 25 mm DTT. B, native gels of PDE5A1 fragments of various lengths. Samples were treated with 25 mm DTT in the presence (+) or absence (−) of 2 mm cGMP. Lanes 1 and 2, PDE5A1-(89–518); lanes 3 and 4, PDE5A1-(98–518); lanes 5 and 6, PDE5A1-(116–518); lanes 7 and 8, PDE5A1-(126–518); lanes 9 and 10, PDE5A1-(142–518).