A conformational switch in the inhibitory gamma-subunit of PDE6 upon enzyme activation by transducin

In response to light, a photoreceptor G protein, transducin, activates cGMP -phosphodiesterase (PDE6) by displacing the inhibitory gamma -subunits (P g amma) from the enzyme's catalytic sites. Evidence suggests that the activat ion of PDE6 involves a conformational change of the key inhibitory C-termin al domain of P gamma. In this study, the C-terminal region of P gamma, P ga mma -73-85, has been targeted for A1a-scanning mutagenesis to identify the point-to-point interactions between P gamma and the PDE6 catalytic subunits and to probe the nature of the conformational change. P gamma mutants were tested for their ability to inhibit PDE6 and a chimeric PDE5-conePDE6 enzy me containing the P gamma C-terminus-binding site of cone PDE. This analysi s has revealed that in addition to previously characterized Ile(86) and Ile (87), important inhibitory contact residues of Py include Asn(74), His(75), and Leu(78). The patterns of mutant PDE5-conePDE6 enzyme inhibition sugges t the interaction between the P gamma ASn(74)/His(75) sequence and Met(758) of the cone PDE6 alpha' catalytic subunit. This interaction, and the inter action between the P gamma Ile(86)/Ile(87) and PDE6 alpha 'Phe(777)/Phe(781 ) residues, is most consistent with an alpha -helical structure of the Py C -terminus. The analysis of activation of PDE6 enzymes containing P gamma mu tants with A1a-substituted transducin-contact residues demonstrated the cri tical role of P gamma Leu(76). Accordingly, we hypothesize that the initial step in PDE6 activation involves an interaction of transducin-alpha with P gamma Leu(76). This interaction introduces a bend into the alpha -helical structure of the P gamma C-terminus, allowing transducin-a to further twist the C-terminus thereby uncovering the catalytic pocket of PDE6.