A highly dynamic active state for transducin-bound phosphodiesterase-6 in vertebrate phototransduction

In vertebrate phototransduction, the G protein-coupled receptor rhodopsin activates the α-subunit of transducin (Gα_T), which, upon binding the γ-subunits of phosphodiesterase-6 (PDE6), stimulates cGMP hydrolysis. We reported a cryoEM structure for a complex containing two constitutively active Gα_T (Gα_T*) subunits coupled by a bivalent antibody bound to PDE6 that demonstrated a striking displacement of both PDEγ subunits from the PDEα/PDEβ catalytic sites and suggested an alternating-site mechanism for PDE6 activation. Here, we use site-directed spin labeling (SDSL) and double electron-electron resonance spectroscopy (DEER) to probe PDE6 conformational changes upon Gα_T* binding. PDEγ spin-labelled on Cys68 and Ile64Cys demonstrate that PDEγ have highly flexible C-termini that transiently bind to the PDEα/PDEβ heterodimer. Binding of Gα_T* to PDE6 with the inhibitor udenafil occupying its catalytic sites alters the positions of the PDEγ subunits in agreement with the changes shown in the cryoEM structure for this complex, whereas coupling the Gα_T* subunits to the bivalent antibody does not affect the DEER distributions observed for PDE6 bound to Gα_T*. However, binding of the slow hydrolyzing 8-Br-cGMP substrate in the presence of Gα_T* causes a dramatic increase in the separation and spread of the spin-labelled PDEγ subunits, thereby revealing a previously unobserved conformation of PDE6 associated with catalysis, which is further supported by small angle X-ray scattering (SAXS) analysis. These studies indicate that whereas inhibitors trap Gα_T*-PDE6 complexes in an inactive state as represented by the cryoEM structure, the binding of both substrate and Gα_T* produces a dynamic active state consistent with an alternating-site mechanism.