Spin-labeling studies implicate a highly dynamic active state for transducin-bound phosphodiesterase-6 in vertebrate phototransduction

In vertebrate phototransduction, the G protein-coupled receptor rhodopsin activates the -subunit of transducin (GT), which, upon binding the {gamma} subunits of phosphodiesterase-6 (PDE6), stimulates the hydrolysis of cGMP. We reported a cryoEM structure for a complex containing two constitutively active GT (GT*) subunits coupled by a bivalent antibody bound to PDE6 that demonstrated a striking displacement of both PDE{gamma} subunits from the PDE/PDE{beta} 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 GT* binding. Both spin-labelled Cys68 on wild-type PDE{gamma} and a spin-labelled cysteine residue substituted for Ile64 on PDE{gamma} demonstrate that PDE{gamma} has highly flexible C-termini that transiently bind to the PDE/PDE{beta} heterodimer. Binding of GT* to PDE6 with the competitive inhibitor udenafil occupying its catalytic sites alters the positions of the PDE{gamma} subunits in agreement with the striking changes shown in the cryoEM structure for this complex, whereas coupling the GT* subunits to the bivalent antibody does not affect the DEER distributions observed for PDE6 bound to GT*. However, binding of the slow hydrolyzing 8-Br-cGMP substrate in the presence of GT* causes a dramatic increase in the separation and spread of the spin-labelled PDE{gamma} subunits, thereby revealing a previously unobserved conformation of PDE6 associated with catalysis. These studies indicate that whereas inhibitors trap GT*-PDE6 complexes in an inactive state as represented by the cryoEM structure, the binding of both substrate and GT* produces a dynamic active state consistent with an alternating site mechanism.