Abstract
The cGMP phosphodiesterase of rod photoreceptor cells, PDE6, is the key effector enzyme in phototransduction. Two large catalytic subunits, PDE6α and -β, each contain one catalytic domain and two non-catalytic GAF domains, whereas two small inhibitory PDE6γ subunits allow tight regulation by the G protein transducin. The structure of holo-PDE6 in complex with the ROS-1 antibody Fab fragment was determined by cryo-electron microscopy. The ∼11 Å map revealed previously unseen features of PDE6, and each domain was readily fit with high resolution structures. A structure of PDE6 in complex with prenyl-binding protein (PrBP/δ) indicated the location of the PDE6 C-terminal prenylations. Reconstructions of complexes with Fab fragments bound to N or C termini of PDE6γ revealed that PDE6γ stretches from the catalytic domain at one end of the holoenzyme to the GAF-A domain at the other. Removal of PDE6γ caused dramatic structural rearrangements, which were reversed upon its restoration.
Highlights
PDE6, the rod photoreceptor phosphodiesterase, is the key effector enzyme in phototransduction
Reconstructions of complexes with Fab fragments bound to N or C termini of PDE6␥ revealed that PDE6␥ stretches from the catalytic domain at one end of the holoenzyme to the GAF-A domain at the other
The pure complexes were examined by SDS-PAGE, concentrated, filtered through a 0.2-m spin-X cellulose acetate filter (Costar), and used directly for cryo-electron microscopy (cryo-EM)
Summary
PDE6, the rod photoreceptor phosphodiesterase, is the key effector enzyme in phototransduction. The ϳ11 Å map revealed previously unseen features of PDE6, and each domain was readily fit with high resolution structures. Reconstructions of complexes with Fab fragments bound to N or C termini of PDE6␥ revealed that PDE6␥ stretches from the catalytic domain at one end of the holoenzyme to the GAF-A domain at the other. The higher resolution map revealed previously unseen features of the PDE6 structure and permitted definitive orientation of the GAF domains. Visualization of antibody Fab fragments bound to the C or N terminus of PDE6␥ was used to investigate the positioning of the inhibitory subunit in the holoenzyme, and imaging of PDE61⁄7PrBP/␦ complexes revealed the locations of the PrBP/␦ binding sites and the C-terminal prenylations of PDE6␣/. Domain Organization and Conformational Plasticity of PDE6 acterized a dramatic structural rearrangement of the catalytic subunits in the absence of PDE6␥
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