Intrinsically disordered γ-subunit of cGMP phosphodiesterase encodes functionally relevant transient secondary and tertiary structure

Abstract

The retinal phosphodiesterase (PDE6) inhibitory ␥-subunit (PDE␥) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic ␣␤-subunits of PDE6 and the ␣-subunit of transducin (␣t). Detailed structural analysis of PDE␥ has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDE␥, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDE␥46-87 when bound to ␣t in the transitionstate complex. NMR mapping of the interaction between PDE␥46-87 and the chimeric PDE5/6 catalytic domain confirmed that Cterminal residues 74 -87 of PDE␥ are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to ␣t, is left free at this stage. These results indicate that the interaction between PDE␥ and ␣t during the phototransduction cascade involves the selection of preconfigured transient conformations.NMR spectroscopy ͉ protein recognition ͉ transient structure ͉ visual transduction P hototransduction, the primary event of vision is critically regulated by the ␥-subunit of cGMP phosphodiesterase (PDE␥). In the dark, by binding tightly to the catalytic ␣␤-subunits of cGMP PDE (PDE6) PDE␥ keeps PDE6 inactive; yet upon photoexcitation, interaction of PDE␥ with the ␣-subunit of activated transducin (␣ t ) relieves its inhibitory constraint on PDE6. For termination of phototransduction, PDE␥ interacts with both ␣ t and RGS9, a regulator of G protein signaling, to accelerate ␣ t GTPase activity (1). Beyond phototransduction, the importance of PDE␥ in photoreceptor cell viability is demonstrated by rapid retinal degeneration in the PDE␥-knockout mouse (2), and growing evidence suggests that PDE␥ also interacts with some signaling proteins in nonphototransduction pathways (3).Biochemical studies have shown that the central polycationic region (residues 24-45) and the C-terminal region of PDE␥ constitute two distinct sites of interactions with ␣ t or PDE6 (4). The crystal structure of a ternary complex representing a partial model for the GTPase-activating protein (GAP) complex, consisting of a fragment of PDE␥ (residues 46-87), ␣ t chimera (␣ t/i1 ), and the catalytic core of RGS9, revealed that the C-terminal region of PDE␥ contains three discontinuous helices (5). PDE␥ 46-87 interacts with ␣ t in a GTP-dependent manner, mainly through residues in these helices or their linkers, and the indole side chain of W70 plays the key role in this interaction. Residue V66 of PDE␥ interacts with RGS9, which further tightens the association between ␣ t and RGS9. The C-terminal region of PDE␥ also interacts with the catalytic domain of PDE6; however, studies have suggested that this interaction involves the 11 C-terminal residues of PDE␥, a region distinct from the ␣ t interaction site (6).It has been demonstrated that PDE␥ belongs to the growing family of intrinsically disordered proteins (IDPs) (7,8). Presumably, the inherent structural plasticity of...

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

“…4) clearly demonstrated that the additional 10 amino acids in the longer truncation mutant induced a partial inhibition of catalytic activity in the absence of the blocking C-terminal segment. This region encompasses the ␣1 helical region previously defined in structural studies (19,20). Furthermore, the ability of the region comprising amino acids 71-76 to further increase the inhibition of cGMP hydrolysis (Fig.…”

“…The proline-rich and polycationic region (amino acids 18 -45, open box) serves as the primary interaction site with the GAF domains of the PDE6 catalytic dimer. The C-terminal domain of P␥ (amino acids ϳ62-87) is shown as consisting of three ␣-helical sequences (␣1, light gray; ␣2, dark gray; ␣3, black) based on structural studies of its complex with transducin ␣-subunit (20) or free in solution (19); the functional interaction of these putative structural elements with PDE6 catalytic dimer is a focus of the experiments in this paper. B, some of the P␥ truncation mutants and synthetic peptides used in this study are depicted.…”

“…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).…”

Mechanism for the allosteric regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-length construct

“…For PDE5, cGMP-induced activation is mediated by binding to the most N-terminal, GAF-A, domain, whereas for PDE2 cGMP interacts with GAF-B, which is more proximal to the catalytic domain. PDE6 is also regulated by cGMP binding to GAF-A, in the unique context of interaction with the P␥ subunit (19,20). The physiological role of GAF domain regulation for PDE10 and 11 is still to be determined (21).…”

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

“…However, the intrinsically disordered PDE␥, especially its N-terminal half (18), has been problematic in solving a crystal structure of ␣t and RGS9⅐␤5 bound with the full-length PDE␥. As indicated in our previous reports (13,19,20), the label transfer approach has allowed for systematic mapping of interactions between fulllength molecules, thus circumventing the problems of intrinsic disorder that can impede efforts in solving atomic structures.…”