The molecular architecture of photoreceptor phosphodiesterase 6 (PDE6) with activated G protein elucidates the mechanism of visual excitation

Abstract

Photoreceptor phosphodiesterase 6 (PDE6) is the central effector of the visual excitation pathway in both rod and cone photoreceptors, and PDE6 mutations that alter PDE6 structure or regulation can result in several human retinal diseases. The rod PDE6 holoenzyme consists of two catalytic subunits (Pαβ) whose activity is suppressed in the dark by binding of two inhibitory γ-subunits (Pγ). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin α-subunit (Gt_α) to PDE6, displacement of Pγ from the PDE6 active site, and enzyme activation. Although the biochemistry of this pathway is understood, a lack of detailed structural information about the PDE6 activation mechanism hampers efforts to develop therapeutic interventions for managing PDE6-associated retinal diseases. To address this gap, here we used a cross-linking MS-based approach to create a model of the entire interaction surface of Pγ with the regulatory and catalytic domains of Pαβ in its nonactivated state. Following reconstitution of PDE6 and activated Gt_α with liposomes and identification of cross-links between Gt_α and PDE6 subunits, we determined that the PDE6-Gt_α protein complex consists of two Gt_α-binding sites per holoenzyme. Each Gt_α interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the Pγ subunit with the catalytic subunits. These results provide the first structural model for the activated state of the transducin-PDE6 complex during visual excitation, enhancing our understanding of the molecular etiology of inherited retinal diseases.

Keywords: G protein; PDE6; allosteric regulation; integrative structural modeling; mass spectrometry (MS); phosphodiesterases; photoreceptor; phototransduction; protein cross-linking; transducin.

Figure 1.

Integrative structural model of the PDE6 holoenzyme. The structural model of rod PDE6 holoenzyme (αβγγ) was determined by using the cryo-EM structure 6MZB (23) as a template and applying spatial restraints determined by chemical cross-linking of purified bovine rod PDE6 (Table 1 and Ref. 20). In the model, PDE6 subunits are colored as follows: α-subunit (Pα), cyan; β-subunit (Pβ), green; Pγ subunit primarily associated with α-subunit (Pγ(Pα)), red; and Pγ subunit primarily associated with β-subunit (Pγ(Pβ)), deep purple. A, superimposition of the template cryo-EM map (EMD-9297) with the cross-link–refined structural model of nonactivated PDE6 holoenzyme. B, asymmetric interactions of Pγ with the Pαβ catalytic dimer extending from the cGMP-binding GAFa domain to the GAFb domain and then crossing over to the catalytic domain to the site of inhibition of catalysis. Each Pγ subunit primarily interacts with one catalytic subunit. The two images are rotated 180°. C, interaction surface of the Pγ(Pα) subunit with the PDE6 catalytic dimer. Pγ(Pα) residues interacting with the catalytic dimer are shown as main-chain atom spheres: red, residues interacting with the α-subunit; pink, residues interacting with the β-subunit; and yellow, Pγ residues that interact with both catalytic subunits. Noninteracting Pγ(Pα) residues are shown as red loops and α-helix. The catalytic subunit interacting residues are shown as a surface representation (α-subunit, dark cyan; β-subunit, dark green). D, interaction surface of the Pγ(Pβ) subunit with Pαβ. The interaction surface of the Pγ(Pβ) subunit (180° rotation in C) is depicted in which the deep purple, light purple, and orange spheres represent interactions with the β-subunit, α-subunit, or both catalytic subunits, respectively.

Figure 2.

Structural model of Gt_α–GDP–AlF₄⁻ and its interaction with Pγ in solution. A, the structural model of Gt_α was determined using the 1TAD crystal structure as the template (36) and refined with spatial restraints imposed from cross-linking results in the absence or presence of Pγ (Table 2). Structural elements that were unchanged in the cross-link–refined model are represented in green, with the conformational change of the αN helix shown in brown (for the crystal structure) and blue (for the cross-link modified solution structure). Also shown is the docked structure of Pγ (red) with Gt_α–GDP–AlF₄⁻ based on the observed cross-linking results when Gt_α associated with lipobeads was incubated with a 2-fold molar excess of Pγ. Note that no significant changes in Gt_α conformation were observed upon Pγ binding. B, a comparison of the conformation of the central region of Pγ (residues 24–44, depicted as a gradient from blue to red spheres) when bound to Gt_α or to the PDE6 catalytic subunits.

Figure 3.

Model of Gt_α–GDP–AlF₄⁻ docked to the Pαβ catalytic dimer. PDE6 holoenzyme and Gt_α–GDP–AlF₄⁻ bound to lipobeads (see “Experimental procedures”) were exposed to chemical cross-linkers, and the identified cross-linked peptides between Gt_α and PDE6 subunits (Table 3) were then used as spatial restraints for integrative structural modeling. Two predominant clusters of models of the Gt_α–Pαβ complex were generated, one with Gt_α docked to the two catalytic domains (with distance violations for Gt_α24-Pα330/Pβ328, Gt_α9-Pα442, and Gt_α9-Pβ440) and the other with Gt_α docked to the GAFb domains (with distance violations for Gt_α10-Pα854, Gt_α17-Pα551, and Gt_α128-Pα807/Pβ817). Because of insufficient cross-links for Pγ in the activated complex, the inhibitory subunit is not shown. A, structural model of association of Gt_α–GDP–AlF₄⁻ to the α-subunit (Gt_α(Pα), orange) and to the β-subunit (Gt_α(Pβ), blue) catalytic domains. B, detailed view of the Gt_α GTPase subdomain interface with the α-subunit catalytic domain, with the interaction surface of Gt_α colored red and the α- and β-subunit interacting residues colored magenta and brown, respectively. The black sphere indicates Gt_α Gln²⁰⁰. C, alternate docking of Gt_α to the GAFb domains of the Pαβ catalytic dimer (with the same orientation as in A).

Figure 4.

Proposed model for the activation of PDE6 by transducin during visual excitation. A, in the dark-adapted condition, the PDE6 holoenzyme is inhibited by its Pγ subunits occluding the enzyme active site (Fig. 1B, rotated 90°). B, the first light-activated Gt_α subunit is proposed to initially bind to the GAFb docking site (see Fig. 3C) without causing significant catalytic activation of PDE6 (21). The Pγ subunit was docked to this complex using the following information: (a) the central region of Pγ (gold) was docked using the cross-links obtained for the Gt_α–Pγ complex (Table 2) in conjunction with the cross-links used to dock Gt_α to the GAFb domain (Table 3); (b) lacking cross-linking data for the N-terminal region of Pγ in the activated complex, this region of Pγ (purple) relied on PDE6 holoenzyme cross-links, and thus its topology only differs from Fig. 1 to the extent needed to accommodate cross-link spatial restraints imposed by the Pγ central region; and (c) in the absence of Pγ cross-links for its C-terminal region in the activated complex, we modeled this region of Pγ (purple) interacting with Gt_α using the crystal structure of Pγ (residues 50–87) bound to a chimeric G protein (PDB code 1FQJ) (11). C, upon binding of a second Gt_α, PDE6 becomes fully activated as both Gt_α subunits dock to the catalytic domains and displace the C-terminal region of Pγ from the enzyme active sites. To accommodate the binding of the central region of Pγ to the helical face (Table 2) and the C-terminal region of Pγ to the GTPase face of Gt_α (Table 3 and Ref. 11), a major displacement of the N-terminal Pγ residues from the GAFa domains must occur.