title: Multiple conformations of phosphodiesterase-5: implications for enzyme function and drug development.
abstract: Phosphodiesterase-5 (PDE5) is the target for sildenafil, vardenafil, and tadalafil, which are drugs for treatment of erectile dysfunction and pulmonary hypertension. We report here the crystal structures of a fully active catalytic domain of unliganded PDE5A1 and its complexes with sildenafil or icarisid II. These structures together with the PDE5A1-isobutyl-1-methylxanthine complex show that the H-loop (residues 660-683) at the active site of PDE5A1 has four different conformations and migrates 7-35A upon inhibitor binding. In addition, the conformation of sildenafil reported herein differs significantly from those in the previous structures of chimerically hybridized or almost inactive PDE5. Mutagenesis and kinetic analyses confirm that the H-loop is particularly important for substrate recognition and that invariant Gly(659), which immediately precedes the H-loop, is critical for optimal substrate affinity and catalytic activity.
authors.affiliation: Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7260.
authors.forename: Huanchen
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7260.
authors.forename: Yudong
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7260.
authors.forename: Qing
authors.initials: Q
authors.lastname: Huai
authors.affiliation: Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7260; School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510080, China.
authors.forename: Jiwen
authors.initials: J
authors.lastname: Cai
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615.
authors.forename: Roya
authors.initials: R
authors.lastname: Zoraghi
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615.
authors.forename: Sharron H
authors.initials: SH
authors.lastname: Francis
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615.
authors.forename: Jackie D
authors.initials: JD
authors.lastname: Corbin
authors.affiliation: Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000.
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.affiliation: Andrology Center, Peking University First Hospital, Peking University, 8 Xishiku Street, Beijing (100034), China.
authors.forename: Zhongcheng
authors.initials: Z
authors.lastname: Xin
authors.affiliation: Department of Urology, University of California, San Francisco, California 94143-1695.
authors.forename: Guiting
authors.initials: G
authors.lastname: Lin
authors.affiliation: Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7260. Electronic address: hke@med.unc.edu.
authors.forename: Hengming
authors.initials: H
authors.lastname: Ke
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry; D000595:Amino Acid Sequence; D002626:Chemistry, Pharmaceutical / Q000379:methods; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D015195:Drug Design; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010879:Piperazines / Q000737:chemistry; D011487:Protein Conformation; D011687:Purines; D017386:Sequence Homology, Amino Acid; D000068677:Sildenafil Citrate; D013379:Substrate Specificity; D013450:Sulfones
chemical_list: D005419:Flavonoids; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011687:Purines; D013450:Sulfones; C060988:baohuoside I; D000068677:Sildenafil Citrate; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human