title: A 46-amino acid segment in phosphodiesterase-5 GAF-B domain provides for high vardenafil potency over sildenafil and tadalafil and is involved in phosphodiesterase-5 dimerization.
abstract: Phosphodiesterase-5 (PDE5) contains a catalytic domain (C domain) that hydrolyzes cGMP and a regulatory domain (R domain) that contains two mammalian cGMP-binding phosphodiesterase, Anabaena adenylyl cyclases, Escherichia coli FhlAs (GAFs) (A and B) and a phosphorylation site for cyclic nucleotide-dependent protein kinases (cNPKs). Binding of cGMP to GAF-A increases cNPK phosphorylation of PDE5 and improves catalytic site affinity for cGMP or inhibitors. GAF-B contributes to dimerization of PDE5, inhibition of cGMP binding to GAF-A, and sequestration of the phosphorylation site. To probe potential PDE5 R domain effects on catalytic site affinity for certain inhibitors, four N-terminal truncation mutants were generated: PDE5Delta1-321 contained GAF-B domain, C domain, and the sequence between GAF-A and -B; PDE5Delta1-419 contained GAF-B and C domain; PDE5Delta1-465 contained the C domain and the C-terminal portion of GAF-B; and PDE5Delta1-534 contained only C domain. Truncated proteins with a complete GAF-B were dimers, but those lacking the N-terminal 46 amino acids of GAF-B were monomers, indicating that these residues are vital for GAF-B-mediated PDE5 dimerization. K(m) values of the mutants for cGMP were similar to that of full-length PDE5. All PDE5 constructs had similar affinities for 3-isobutyl-1-methylxanthine, sildenafil, tadalafil, and UK-122764, but mutants containing a complete GAF-B had 7- to 18-fold higher affinity for vardenafil-based compounds compared with those lacking a complete GAF-B. This indicated that the N-terminal 46 amino acids in GAF-B are required for high vardenafil potency. This is the first evidence that PDE5 R domain, and GAF-B in particular, influences affinity and selectivity of the catalytic site for certain classes of inhibitors.
authors.affiliation: Department of Molecular Physiology and Biophysics, 702 Light Hall, Vanderbilt University School of Medicine, Nashville, TN 37232-0615, USA.
authors.forename: Mitsi A
authors.initials: MA
authors.lastname: Blount
authors.forename: Roya
authors.initials: R
authors.lastname: Zoraghi
authors.forename: Hengming
authors.initials: H
authors.lastname: Ke
authors.forename: Emmanuel P
authors.initials: EP
authors.lastname: Bessay
authors.forename: Jackie D
authors.initials: JD
authors.lastname: Corbin
authors.forename: Sharron H
authors.initials: SH
authors.lastname: Francis
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002243:Carbolines / Q000737:chemistry / Q000494:pharmacology*; D002499:Centrifugation, Density Gradient; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D019281:Dimerization; D020035:Holoenzymes / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008970:Molecular Weight; D050505:Mutant Proteins / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry; D010879:Piperazines / Q000737:chemistry / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D011687:Purines; D000068677:Sildenafil Citrate; D013329:Structure-Activity Relationship; D013450:Sulfones / Q000737:chemistry / Q000494:pharmacology; D000068581:Tadalafil; D014227:Triazines / Q000737:chemistry / Q000494:pharmacology; D014316:Tritium; D000069058:Vardenafil Dihydrochloride
chemical_list: D002243:Carbolines; D020035:Holoenzymes; D007093:Imidazoles; D050505:Mutant Proteins; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D013450:Sulfones; D014227:Triazines; D014316:Tritium; D000069058:Vardenafil Dihydrochloride; D000068581:Tadalafil; D000068677:Sildenafil Citrate; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human