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

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 (Palphabeta) whose activity is suppressed in the dark by binding of two inhibitory gamma-subunits (Pgamma). Upon photoactivation of rhodopsin, the heterotrimeric G protein (transducin) is activated, resulting in binding of the activated transducin alpha-subunit (Gtalpha) to PDE6, displacement of Pgamma 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 Pgamma with the regulatory and catalytic domains of Palphabeta in its nonactivated state. Following reconstitution of PDE6 and activated Gtalpha with liposomes and identification of cross-links between Gtalpha and PDE6 subunits, we determined that the PDE6-Gtalpha protein complex consists of two Gtalpha-binding sites per holoenzyme. Each Gtalpha interacts with the catalytic domains of both catalytic subunits and induces major changes in the interaction sites of the Pgamma 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.