Structural basis of phosphodiesterase 6 inhibition by the C-terminal region of the γ-subunit

Abstract

The inhibitory interaction of phosphodiesterase-6 (PDE6) with its c-subunit (Pc) is pivotal in vertebrate phototransduction. Here, crystal structures of a chimaeric PDE5/ PDE6 catalytic domain (PDE5/6cd) complexed with sildenafil or 3-isobutyl-1-methylxanthine and the Pc-inhibitory peptide Pc 70À87 have been determined at 2.9 and 3.0 Å , respectively. These structures show the determinants and the mechanism of the PDE6 inhibition by Pc and suggest the conformational change of Pc on transducin activation. Two variable H-and M-loops of PDE5/6cd form a distinct interface that contributes to the Pc-binding site. This allows the Pc C-terminus to fit into the opening of the catalytic pocket, blocking cGMP access to the active site. Our analysis suggests that disruption of the H-M loop interface and Pc-binding site is a molecular cause of retinal degeneration in atrd3 mice. Comparison of the two PDE5/6cd structures shows an overlap between the sildenafil and Pc 70À87 -binding sites, thereby providing critical insights into the side effects of PDE5 inhibitors on vision.

Structural Requirements of the Photoreceptor Phosphodiesterase γ-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

“…Our functional definition of the blocking region of P␥ in this study (Fig. 7, region B) is consistent with structural studies revealing that P␥ residues 78 -87 assume a nearly identical conformation when bound to the ␣-subunit of transducin (20) or when complexed with a PDE5/6 chimeric catalytic domain (21).…”

“…Structural studies of P␥ in solution indicate that this protein is overall an intrinsically disordered protein (18) but contains ␣-helical secondary structure (␣1, ␣2, and ␣3; Fig. 1A) in its C-terminal region (19); the ␣-helical content within the C-terminal region of P␥ is also observed when this region of P␥ forms a complex with either transducin (20) or a chimeric PDE5/6 catalytic domain (21).…”

Functional Mapping of Interacting Regions of the Photoreceptor Phosphodiesterase (PDE6) γ-Subunit with PDE6 Catalytic Dimer, Transducin, and Regulator of G-protein Signaling9–1 (RGS9–1)

“…7). We hypothesize that this stretch of amino acids stabilizes binding of T␣* to position it to develop additional interactions with the C-terminal "hinge" (amino acids 71-77) and "blocking" (amino acids 78 -87) regions of P␥ (8,24) that lead to de-inhibition of PDE6 catalysis. A subset of residues within the glycine-rich region of P␥ (specifically amino acids 55-62) is also implicated in facilitating the potentiating role of P␥ to accelerate GTPase activity of the T␣*/RGS9 -1 complex.…”

“…The primary regulatory role of P␥ is to regulate access of substrate to the catalytic pocket of PDE6 and thereby control cGMP hydrolytic rates. This function is carried out by the last few C-terminal residues of P␥ interacting with the PDE6 catalytic domains in the immediate vicinity of the active site (7)(8)(9)). An allosterically mediated inhibition of catalysis that occurs in the absence of the C-ter-minal residues of P␥ has also been identified (10).…”

N-terminal Half of the cGMP Phosphodiesterase γ-Subunit Contributes to Stabilization of the GTPase-accelerating Protein Complex

“…In the partial GAP structure (7), however, only one direct contact between PDE␥ and RGS9d has been discovered. It is therefore important to explore whether the PDE␥ N-terminal half, which is absent in the existing crystal structures (7,15,17), also plays a role in the GAP complex by interacting with RGS9⅐␤5.…”