title: Direct allosteric regulation between the GAF domain and catalytic domain of photoreceptor phosphodiesterase PDE6.
abstract: Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory gamma subunit (Pgamma), known to directly inhibit PDE6 catalysis, was increased approximately 2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of Pgamma to the GAF domains suffices to induce this allosteric effect. Allosteric communication between GAF and catalytic domains is reciprocal, in that drug binding to the catalytic domain slowed cGMP dissociation from the GAF domain. Although cGMP hydrolysis was not affected by binding of Pgamma1-60, Pgamma lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5-fold. Pgamma1-60 binding to the GAF domain increased vardenafil but not cGMP affinity, indicating that substrate- and inhibitor-binding sites do not totally overlap. In addition, prolonged incubation of PDE6 with vardenafil or sildenafil (but not 3-isobutyl-1-methylxanthine and zaprinast) induced a distinct conformational change in the catalytic domain without affecting the binding properties of the GAF domains. We conclude that although Pgamma-mediated regulation plays the dominant role in visual excitation, the direct, inter-domain allosteric regulation described in this study may play a feedback role in light adaptational processes during phototransduction.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824, USA.
authors.forename: Xiu-Jun
authors.initials: XJ
authors.lastname: Zhang
authors.forename: Karyn B
authors.initials: KB
authors.lastname: Cahill
authors.forename: Arye
authors.initials: A
authors.lastname: Elfenbein
authors.forename: Vadim Y
authors.initials: VY
authors.lastname: Arshavsky
authors.forename: Rick H
authors.initials: RH
authors.lastname: Cote
mesh_terms: D000495:Allosteric Site; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6 / Q000737:chemistry / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D007093:Imidazoles / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008968:Molecular Conformation; D010879:Piperazines / Q000494:pharmacology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011687:Purines / Q000494:pharmacology; D012160:Retina / Q000378:metabolism; D000068677:Sildenafil Citrate; D013450:Sulfones / Q000494:pharmacology; D014227:Triazines / Q000494:pharmacology; D000069058:Vardenafil Dihydrochloride
chemical_list: D007093:Imidazoles; D008024:Ligands; D010879:Piperazines; D011687:Purines; D013450:Sulfones; D014227:Triazines; D000069058:Vardenafil Dihydrochloride; D000068677:Sildenafil Citrate; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D006152:Cyclic GMP
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