Structural characterization of the rod cGMP phosphodiesterase 6.
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.
Goc
Anna
A
Department of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4965, USA.
Chami
Mohamed
M
Lodowski
David T
DT
Bosshart
Patrick
P
Moiseenkova-Bell
Vera
V
Baehr
Wolfgang
W
Engel
Andreas
A
Palczewski
Krzysztof
K
D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D006152:Cyclic GMP; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6 / Q000737:chemistry* / Q000302:isolation & purification*; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D054876:Prenylation; D011487:Protein Conformation; D021122:Protein Subunits; D012374:Rod Cell Outer Segment / Q000201:enzymology*
D010446:Peptide Fragments; D021122:Protein Subunits; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D006152:Cyclic GMP