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 (P gamma), known to directly inhibit PDE6 catalysis, was increased similar to 2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of P gamma 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 P gamma-60, P gamma lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5- fold. P gamma 1-60 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 P gamma-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.

Publication Date

10-31-2008

Journal Title

Journal of Biological Chemistry

Publisher

American Society for Biochemistry and Molecular Biology

Digital Object Identifier (DOI)

10.1074/jbc.M803948200

Scientific Contribution Number

2361

Document Type

Article

Rights

© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.