Probing the mechanism by which the retinal G protein transducin activates its biological effector PDE6

Abstract

Phototransduction in retinal rods occurs when the G protein-coupled photoreceptor rhodopsin triggers the activation of phosphodiesterase 6 (PDE6) by GTP-bound alpha subunits of the G protein transducin (Gα_T). Recently, we presented a cryo-EM structure for a complex between two GTP-bound recombinant Gα_T subunits and native PDE6, that included a bivalent antibody bound to the C-terminal ends of Gα_T and the inhibitor vardenafil occupying the active sites on the PDEα and PDEβ subunits. We proposed Gα_T-activated PDE6 by inducing a striking reorientation of the PDEγ subunits away from the catalytic sites. However, questions remained including whether in the absence of the antibody Gα_T binds to PDE6 in a similar manner as observed when the antibody is present, does Gα_T activate PDE6 by enabling the substrate cGMP to access the catalytic sites, and how does the lipid membrane enhance PDE6 activation? Here, we demonstrate that 2:1 Gα_T-PDE6 complexes form with either recombinant or retinal Gα_T in the absence of the Gα_T antibody. We show that Gα_T binding is not necessary for cGMP nor competitive inhibitors to access the active sites; instead, occupancy of the substrate binding sites enables Gα_T to bind and reposition the PDE6γ subunits to promote catalytic activity. Moreover, we demonstrate by reconstituting Gα_T-stimulated PDE6 activity in lipid bilayer nanodiscs that the membrane-induced enhancement results from an increase in the apparent binding affinity of Gα_T for PDE6. These findings provide new insights into how the retinal G protein stimulates rapid catalytic turnover by PDE6 required for dim light vision.

Keywords: G protein; cryoelectron microscopy; phosphodiesterase; phototransduction; signal transduction; structural biology.