Molecular determinants of cGMP binding to chicken cone photoreceptor phosphodiesterase.
Structural studies on photoreceptor phosphodiesterases type 6 (PDE6s) have been hampered by an inability to express and purify substantial amounts of enzyme. Here we describe bacterial expression and characterization of the chicken cone PDE6 regulatory GAF-A and GAF-B domains. High affinity cGMP binding was found only for GAF-A as predicted from sequence alignments with the GAF domains of PDE2 and PDE5. A homology model of the GAF-A domain of chicken cone PDE6 based on the crystal structure of mouse PDE2A GAF-B was used to identify residues likely to make contact with cGMP. Alanine mutagenesis of 4 of these residues (F123A, D169A, T172A, and T176A) showed that each was absolutely required for cGMP binding. Three of these residues map to the H4 helical structure of the GAF-A domain indicating this region as a key structural component for cGMP binding. Mutagenesis of another residue, S97A, decreased cGMP binding affinity 5-fold. Finally mutagenesis of Glu-124 indicated that it is responsible for part but not all of the high specificity for cGMP binding to PDE6 GAF-A. Since little data is available on the properties of the chicken cone PDE6 holoenzyme, we also characterized the native PDEs of chicken retina. Two histone-activated PDE6 peaks were separated by ion exchange chromatography and identified by mass spectrometry as cone and rod photoreceptor PDE6s, respectively. Both of these PDEs had cGMP binding and kinetic properties similar to their corresponding bovine photoreceptor PDEs. Moreover the cGMP binding properties of chicken cone PDE6 holoenzyme were very similar to those of the bacterially expressed individual GAF-A or GAF-A/B domains.
Huang
Daming
D
Department of Pharmacology and Medicinal Chemistry, University of Washington, Seattle, Washington 98195, USA.
Hinds
Thomas R
TR
Martinez
Sergio E
SE
Doneanu
Catalin
C
Beavo
Joseph A
JA
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D006152:Cyclic GMP / Q000378:metabolism*; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000201:enzymology*; D016415:Sequence Alignment
D005136:Eye Proteins; D021122:Protein Subunits; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054678:Cyclic Nucleotide Phosphodiesterases, Type 2; C517277:Pde2a protein, mouse; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP