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 (Gtα) 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 Gtα with liposomes and identification of cross-links between Gtα and PDE6 subunits, we determined that the PDE6-Gtα protein complex consists of two Gtα-binding sites per holoenzyme. Each Gtα 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.
Highlights
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
Upon comparing the 3.4 Å cryo-EM structure of the PDE6 holoenzyme [23] with our previous solution structure of PDE6 catalytic dimer determined by chemical cross-linking, identification of cross-linked peptides by mass spectrometric analysis, and integrative structural modeling [20], we observed that a number of distance restraints defined by our cross-linking results were inconsistent with the cryo-EM structure
This paper reports the first complete structural models for the PDE6 holoenzyme (Fig. 1), the activated ␣-subunit of transducin in a complex with the inhibitory ␥-subunit of PDE6 (Fig. 2), and the fully activated state of PDE6 in a complex with two transducin ␣-subunits (Fig. 3)—all in their membrane-associated state that mimics the localization of the transducin–PDE6 protein complex on photoreceptor outer segment disk membranes
Summary
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.
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