Structural characterization of the rod cGMP phosphodiesterase 6

Abstract

Rod cGMP phosphodiesterase 6 (PDE6) is a key enzyme of the phototransduction cascade, consisting of PDE6alpha, PDE6beta, and two regulatory PDE6gamma subunits. PDE6 is membrane associated through isoprenyl membrane anchors attached to the C-termini of PDE6alpha and PDE6beta and can form a complex with prenyl-binding protein delta (PrBP/delta), an isoprenyl-binding protein that is highly expressed in photoreceptors. The stoichiometry of PDE6-PrBP/delta binding and the mechanism by which the PDE6-PrBP/delta complex assembles have not been fully characterized, and the location of regulatory PDE6gamma subunits within the protein assembly has not been elucidated. To clarify these questions, we have developed a rapid purification method for PDE6-PrBP/delta from bovine rod outer segments utilizing recombinant PrBP/delta. Transmission electron microscopy of negatively stained samples revealed the location of PrBP/delta and, thus, where the carboxyl-termini of PDE6alpha and PDE6beta must be located. The three-dimensional structure of the PDE6alphabetagamma complex was determined up to 18 A resolution from single-particle projections and was interpreted by model building to identify the probable location of isoprenylation, PDE6gamma subunits, and catalytic sites.