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 (Pgamma) from the enzyme's catalytic sites. Evidence suggests that the activation of PDE6 involves a conformational change of the key inhibitory C-terminal domain of Pgamma. In this study, the C-terminal region of Pgamma, Pgamma-73-85, has been targeted for Ala-scanning mutagenesis to identify the point-to-point interactions between Pgamma and the PDE6 catalytic subunits and to probe the nature of the conformational change. Pgamma mutants were tested for their ability to inhibit PDE6 and a chimeric PDE5-conePDE6 enzyme containing the Pgamma C-terminus-binding site of cone PDE. This analysis has revealed that in addition to previously characterized Ile86 and Ile87, important inhibitory contact residues of Pgamma include Asn74, His75, and Leu78. The patterns of mutant PDE5-conePDE6 enzyme inhibition suggest the interaction between the PgammaAsn74/His75 sequence and Met758 of the cone PDE6alpha' catalytic subunit. This interaction, and the interaction between the PgammaIle86/Ile87 and PDE6alpha'Phe777/Phe781 residues, is most consistent with an alpha-helical structure of the Pgamma C-terminus. The analysis of activation of PDE6 enzymes containing Pgamma mutants with Ala-substituted transducin-contact residues demonstrated the critical role of PgammaLeu76. Accordingly, we hypothesize that the initial step in PDE6 activation involves an interaction of transducin-alpha with PgammaLeu76. This interaction introduces a bend into the alpha-helical structure of the Pgamma C-terminus, allowing transducin-alpha to further twist the C-terminus thereby uncovering the catalytic pocket of PDE6.
Granovsky
A E
AE
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Artemyev
N O
NO
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry* / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002417:Cattle; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D020419:Photoreceptor Cells, Vertebrate / Q000201:enzymology; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D021122:Protein Subunits; D015293:Transducin / Q000494:pharmacology*
D010446:Peptide Fragments; D021122:Protein Subunits; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin; D006152:Cyclic GMP; D000409:Alanine