Abstract
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 the hydrolysis of cGMP. 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. Both spin-labelled Cys68 on wild-type PDEγ and a spin-labelled cysteine residue substituted for Ile64 on PDEγ demonstrate that PDEγ has highly flexible C-termini that transiently bind to the PDEα/PDEβ heterodimer. Binding of Gα(T)* to PDE6 with the competitive inhibitor udenafil occupying its catalytic sites alters the positions of the PDEγ subunits in agreement with the striking 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. 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.