FUNCTIONAL AND MUTATIONAL ANALYSIS OF THE IDENTIFICATION OF Pγ-BINDING RESIDUES ON PDE6α'

Abstract

Photoreceptor cGMP phosphodiesterase (PDE 6) is an effector enzyme in the G-protein mediated visual transduction cascade. In the dark, PDE6 activity is turned off by an inhibitory γ- subunit (Pγ). To study the mechanism of PDE6 catalytic activity inhibition by Pγ chimeric proteins PDE6α’/PDE5 were created. The catalytic properties of chimeric PDE remained the same as those of PDE5. Mutational analysis of the Pg-binding region, PDE6α' - (750-760), using ala-scan revealed PDE6α’ residues required for interaction. The M758A mutation markedly impaired and the Q752A mutation moderately impaired Pγ inhibition of the chimeric PDE. Analysis of the catalytic properties of the mutant PDE and the PDE6 catalytic domain model suggest that the Met758 and Gln752 residues bind Pγ directly. The PDE6 catalytic site model shows that PDE6α’-(750–760) forms a loop at the entrance to the cGMP-binding pocket. Binding of Pγ to Met758 effectively blocks cGMP access to the catalytic cavity, providing a structural basis for the PDE6 inhibition mechanism.