Portal de Periódicos da CAPES

Multiple Conformations of Phosphodiesterase-5

2006; Elsevier BV; Volume: 281; Issue: 30 Linguagem: Inglês

10.1074/jbc.m512527200

1083-351X

Huanchen Wang, Yudong Liu, Qing Huai, Jiwen Cai, Roya Zoraghi, Sharron H. Francis, Jackie D. Corbin, Howard Robinson, Zhongcheng Xin, Guiting Lin, Hengming Ke,

Chemical synthesis and alkaloids

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 7-35Å 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 Gly659, which immediately precedes the H-loop, is critical for optimal substrate affinity and catalytic activity. 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 7-35Å 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 Gly659, which immediately precedes the H-loop, is critical for optimal substrate affinity and catalytic activity. Cyclic nucleotide phosphodiesterases (PDEs) 3The abbreviations used are: PDEs, phosphodiesterases; IBMX, 3-isobutyl-1-methylxanthine; r.m.s., root mean square. 3The abbreviations used are: PDEs, phosphodiesterases; IBMX, 3-isobutyl-1-methylxanthine; r.m.s., root mean square.are key enzymes controlling cellular concentrations of second messengers cAMP and cGMP by hydrolyzing them to 5′-AMP and 5′-GMP, respectively. The human genome encodes 21 PDE genes that are categorized into 11 families (1Manganiello V.C. Taira M. Degerman F. Belfrage P. Cell. Signal. 1995; 7: 445-455Crossref PubMed Scopus (119) Google Scholar, 2Houslay M.D. Sullivan M. Bolger G.B. Adv. Pharmacol. 1998; 44: 225-343Crossref PubMed Scopus (282) Google Scholar, 3Torphy T.J. Am. J. Respir. Crit. Care Med. 1998; 157: 351-370Crossref PubMed Scopus (656) Google Scholar, 4Soderling S.H. Beavo J.A. Curr. Opin. Cell Biol. 2000; 12: 174-179Crossref PubMed Scopus (645) Google Scholar, 5Francis S.H. Turko I.V. Corbin J.D. Prog. Nucleic Acids Res. Mol. Biol. 2001; 65: 1-52Crossref PubMed Google Scholar, 6Mehats C. Andersen C.B. Filopanti M. Jin S.L. Conti M. Trends Endocrinol. Metab. 2002; 13: 29-35Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 7Conti M. Richter W. Mehats C. Livera G. Park J.Y. Jin C. J. Biol. Chem. 2003; 278: 5493-5496Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 8Houslay M.D. Adams D.R. Biochem. J. 2003; 370: 1-18Crossref PubMed Scopus (646) Google Scholar, 9Goraya T.A. Cooper D.M. Cell. Signal. 2005; 17: 789-797Crossref PubMed Scopus (95) Google Scholar). Alternative mRNA splicing of the PDE genes produces over 60 PDE isoforms that distribute in various cellular compartments and control physiological processes. PDE molecules contain a conserved catalytic domain and variable regulatory regions. However, each PDE family possesses a characteristic pattern of substrate specificity and inhibitor selectivity (6Mehats C. Andersen C.B. Filopanti M. Jin S.L. Conti M. Trends Endocrinol. Metab. 2002; 13: 29-35Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). Inhibitors of PDEs have been widely studied as therapeutics as follows: cardiotonics, vasodilators, smooth muscle relaxants, antidepressants, antithrombotics, antiasthmatics, and agents for improving cognitive functions such as learning and memory (10Movsesian M.A. Exp. Opin. Investig. Drugs. 2000; 9: 963-973Crossref PubMed Scopus (22) Google Scholar, 11Truss M.C. Stief C.G. Uckert S. Becker A.J. Wafer J. Schultheiss D. Jonas U. World J. Urol. 2001; 19: 344-350Crossref PubMed Scopus (81) Google Scholar, 12Liu Y. Shakur Y. Yoshitake M. Kambayashi J.J. Cardiovasc. Drug Rev. 2001; 19: 369-386Crossref PubMed Scopus (173) Google Scholar, 13Huang Z. Ducharme Y. MacDonald D. Robinchaud A. Curr. Opin. Chem. Biol. 2001; 5: 432-438Crossref PubMed Scopus (124) Google Scholar, 14Rotella D.P. Nat. Rev. Drug Discovery. 2002; 1: 674-682Crossref PubMed Scopus (260) Google Scholar, 15Corbin J.D. Francis S.H. Int. J. Clin. Pract. 2002; 56: 453-459PubMed Google Scholar, 16Lipworth B.J. Lancet. 2005; 365: 167-175Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 17Castro A. Jerez M.J. Gil C. Martinez A. Med. Res. Rev. 2005; 25: 229-244Crossref PubMed Scopus (105) Google Scholar). Some of the most successful examples of these drugs are the PDE5 inhibitors sildenafil (Viagra®), vardenafil (Levitra®), and tadalafil (Cialis®) that have been used for treatment of male erectile dysfunction (15Corbin J.D. Francis S.H. Int. J. Clin. Pract. 2002; 56: 453-459PubMed Google Scholar). Sildenafil has also recently been approved (Revatio®) for treatment of pulmonary hypertension (18Galie N. Ghofrani H.A. Torbicki A. Barst R.J. Rubin L.J. Badesch D. Fleming T. Parpia T. Burgess G. Branzi A. Grimminger F. Kurzyna M. Simonneau G. N. Engl. J. Med. 2005; 353: 2148-2157Crossref PubMed Scopus (2041) Google Scholar). However, reported side effects of these medications such as headache and visual disturbance suggest a need for further study of the molecular basis of the selectivity of PDE5 inhibitors (19Pomeranz H.D. Bhavsar A.R. J. Neuroophthalmol. 2005; 25: 9-13Crossref PubMed Scopus (140) Google Scholar). Two co-crystal structures of the catalytic domain of PDE5 with sildenafil showed differences in the conformation of the inhibitor bound to the catalytic site (20Sung B.J. Hwang K.Y. Jeon Y.H. Lee J.I. Heo Y.S. Kim J.H. Moon J. Yoon J.M. Hyun Y.L. Kim E. Eum S.J. Park S.Y. Lee J.O. Lee T.G. Ro S. Cho J.M. Nature. 2003; 425: 98-102Crossref PubMed Scopus (233) Google Scholar, 21Brown D.G. Groom C.R. Hopkins A.L. Jenkins T.M. Kamp S.H. O'Gara M.M. Ringrose H.J. Robinson C.M. Taylor W.E. WIPO Patent.WO 03/2003/038080. May 8 2003; Google Scholar, 22Zhang K.Y. Card G.L. Suzuki Y. Artis D.R. Fong D. Gillette S. Hsieh D. Neiman J. West B.L. Zhang C. Milburn M.V. Kim S.H. Schlessinger J. Bollag G. Mol. Cell. 2004; 15: 279-2861Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). However, it remains unknown whether these conformations are biologically relevant because the PDE5 enzyme used in the studies is either almost inactive (20Sung B.J. Hwang K.Y. Jeon Y.H. Lee J.I. Heo Y.S. Kim J.H. Moon J. Yoon J.M. Hyun Y.L. Kim E. Eum S.J. Park S.Y. Lee J.O. Lee T.G. Ro S. Cho J.M. Nature. 2003; 425: 98-102Crossref PubMed Scopus (233) Google Scholar) or a chimeric hybrid of the PDE5 catalytic domain with replacement of a PDE4 segment (the H-loop) (21Brown D.G. Groom C.R. Hopkins A.L. Jenkins T.M. Kamp S.H. O'Gara M.M. Ringrose H.J. Robinson C.M. Taylor W.E. WIPO Patent.WO 03/2003/038080. May 8 2003; Google Scholar, 22Zhang K.Y. Card G.L. Suzuki Y. Artis D.R. Fong D. Gillette S. Hsieh D. Neiman J. West B.L. Zhang C. Milburn M.V. Kim S.H. Schlessinger J. Bollag G. Mol. Cell. 2004; 15: 279-2861Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). In addition, the crystal structure of the catalytic domain of PDE5A1 in complex with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) showed that the conformation of the H-loop at the active site is different from that in PDE4 (23Huai Q. Liu Y. Francis S.H. Corbin J.D. Ke H. J. Biol. Chem. 2004; 279: 13095-13101Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and in other published PDE5 structures (20Sung B.J. Hwang K.Y. Jeon Y.H. Lee J.I. Heo Y.S. Kim J.H. Moon J. Yoon J.M. Hyun Y.L. Kim E. Eum S.J. Park S.Y. Lee J.O. Lee T.G. Ro S. Cho J.M. Nature. 2003; 425: 98-102Crossref PubMed Scopus (233) Google Scholar, 21Brown D.G. Groom C.R. Hopkins A.L. Jenkins T.M. Kamp S.H. O'Gara M.M. Ringrose H.J. Robinson C.M. Taylor W.E. WIPO Patent.WO 03/2003/038080. May 8 2003; Google Scholar, 22Zhang K.Y. Card G.L. Suzuki Y. Artis D.R. Fong D. Gillette S. Hsieh D. Neiman J. West B.L. Zhang C. Milburn M.V. Kim S.H. Schlessinger J. Bollag G. Mol. Cell. 2004; 15: 279-2861Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 24Card G.L. England B.P. Suzuki Y. Fong D. Powell B. Lee B. Luu C. Tabrizizad M. Gillette S. Ibrahim P.N. Artis D.R. Bollag G. Milburn M.V. Kim S.H. Schlessinger J. Zhang K.Y. Structure (Camb.). 2004; 12: 2233-2247Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). We report here the structure of the catalytic domain of human PDE5A1 in the unliganded state, as well as the structures of the protein in complex with inhibitors sildenafil and icarisid II (Fig. 1). These structures, together with that of PDE5A1-IBMX, reveal four different conformations of the H-loop, which is juxtaposed to the catalytic pocket of the enzyme. In addition, comparison of this PDE5-sildenafil structure with the previously published structures shows significantly different conformations of the methylpiperazine portion of sildenafil. These unique features of the PDE5 catalytic domain and the sildenafil configuration are key considerations for understanding the action of sildenafil and for development of PDE5 inhibitors. Protein Expression and Purification of Catalytic Domain PDE5A1—The cDNA of the catalytic domain of human PDE5A1 was generated by site-directed mutagenesis of the bovine PDE5A cDNA (23Huai Q. Liu Y. Francis S.H. Corbin J.D. Ke H. J. Biol. Chem. 2004; 279: 13095-13101Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). The coding regions for amino acids 535-860 of PDE5A1 were amplified by PCR and subcloned into the expression vector pET15b. The resultant plasmid pET-PDE5A1 was transferred into Escherichia coli strain BL21 (Codonplus) for overexpression. The E. coli cell carrying pET-PDE5A1 was grown in LB medium at 37 °C to an A600 = 0.7, and 0.1 mm isopropyl β-d-thiogalactopyranoside was then added for further growth at 15 °C overnight. Recombinant PDE5A1 was purified by the columns of nickel-nitrilotriacetic acid (Qiagen), Q-Sepharose, and Sephacryl S300 (Amersham Biosciences). A typical purification yielded over 10 mg of PDE5A1 with a purity of >95% from a 2-liter cell culture. The mutant proteins with deletion of residues 663-678 and 661-681 were produced by the standard protocol of site-directed mutagenesis. For the deletion mutant proteins, four glycine residues were inserted as the spacer to minimize the disturbance on the three-dimensional structure. Overexpression and purification of the mutant proteins used the same protocols as for the nonmutated protein. Expression and Purification of Full-length hPDE5A1—Human cDNA coding for full-length PDE5A1 was cloned into pCR 2.1-TOPO® vector (Invitrogen) and then ligated into the baculovirus expression vector pAcHLT-A (Pharmingen). The resulting plasmid pAcA-PDE5 (Met1-Asn875) was used to make point mutations (G659A, V660Q, N661A, N662A, Y664A, H678A, and S679A) with the QuikChange site-directed mutagenesis kit (Stratagene). Wild type and mutant constructs of hPDE5A1 were expressed in Sf9 cells. Sf9 cells were cotransfected with BaculoGold linear baculovirus DNA (Pharmingen) and one of the pAcA-hPDE5A1 plasmids. The cotransfection supernatant was collected at 5 days post-infection, amplified three times in Sf9 cells, and then used directly as virus stock without additional purification. Sf9 cells grown at 27 °C in complete Grace's insect medium with 10% fetal bovine serum and 10 μg/ml gentamicin (Sigma) were typically infected with 100 μl of viral stock and then harvested at 92 h post-infection. Protein yields were ∼2.7 mg/liter infected cell media and were largely soluble. The Sf9 cell pellet (∼2 × 107 cells) was resuspended in icecold lysis buffer (20 mm Tris-HCl, pH 8, 100 mm NaCl) containing protease inhibitor mixture, homogenized, and centrifuged. The supernatant was loaded onto a nickel-nitrilotriacetic acidagarose (Qiagen) column equilibrated with lysis buffer. The column was washed with lysis buffer containing a stepwise gradient of 0.8-20 mm imidazole and eluted with 100 mm imidazole. Eluted PDE5A1 protein was dialyzed versus 2000 volumes of ice-cold 10 mm potassium phosphate, pH 6.8, 25 mm β-mercaptoethanol, and 150 mm NaCl. All recombinant PDE5A1 proteins exhibited a purity of >98% as determined by SDS-PAGE. Enzymatic Assay—Enzymatic activity of the isolated catalytic domains of wild type PDE5A1 and its deletion mutants was assayed by using [3H]cGMP as substrate in a reaction mixture of 20 mm Tris-HCl, pH 7.8, 1.5 mm dithiothreitol, 10 mm MgCl2,[3 H]cGMP (40,000 cpm/assay) at 24 °C for 15 min (25Wang H. Liu Y. Chen Y. Robinson H. Ke H. J. Biol. Chem. 2005; 280: 30949-30955Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The reaction was terminated by addition of 0.2 m ZnSO4 and Ba(OH)2. Radioactivity of unreacted [3 H]cGMP in the supernatant was measured by a liquid scintillation counter. The turnover rate was measured at nine concentrations of cGMP and controlled at hydrolysis of 15-40% substrate. Each measurement was repeated three times. For measurement of IC50 values, 10 concentrations of inhibitors were used at a substrate concentration that was one-tenth of the Km value and an enzyme concentration that hydrolyzed 50% of substrate. To assay the activity of full-length hPDE5A1, the reaction mixture contained 50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 0.3 mg/ml bovine serum albumin, 0.05-250 μm cGMP, and [3H]cGMP (100,000-150,000 cpm/assay) and one of the PDE5A1 proteins (26Thomas M.K. Francis S.H. Corbin J.D. J. Biol. Chem. 1990; 265: 14971-14978Abstract Full Text PDF PubMed Google Scholar). Incubation time was 10 min at 30 °C. In all studies, less than 10% of total [3H]cGMP was hydrolyzed during the reaction. All values determined represent three measurements. The Km and kcat values for cGMP were determined by nonlinear regression analysis of data using Prism GraphPad software. Crystallization and Data Collection—All crystals of PDE5A1-(535-860) were grown by vapor diffusion. The protein drop was prepared by mixing 2 μl of protein with 2 μl of well buffer. The unliganded PDE5A1 crystal was grown at room temperature against well buffer of 0.2 m MgSO4, 0.1 m Tris base, pH 8.5, 12% PEG 3350, and 2% ethanol. The PDE5A1-sildenafil complex was prepared by mixing 1 mm sildenafil with 15 mg/ml PDE5A1 at 4 °C overnight and crystallized against a well buffer of 1.0 m sodium citrate, 2.5% ethanol, 0.1 m HEPES, pH 7.5, at 4 °C. The PDE5A1-icarisid II complex was prepared by mixing 2 mm icarisid II with 15 mg/ml protein at 4 °C overnight, and crystallized against a well buffer of 0.1 m HEPES, pH 7.5, 12% PEG3350 at room temperature. The unliganded PDE5A1-(535-860) was crystallized in the space group P3121 with cell dimensions of a = b = 74.7 and c = 130.7 Å. The PDE5A1-sildenafil crystal had the space group P6222 with cell dimensions of a = b = 164.6 and c = 193.1 Å. The PDE5A1-icarisid II crystal had the space group P6122 with cell dimensions of a = b = 110.7 and c = 106.2 Å. Beamline X25 at Brookhaven National Laboratory was used for collection of diffraction data of the unliganded PDE5A1 and X29 for PDE5A1-sildenafil and PDE5A1-icarisid II (Table 1). All data were processed by the program HKL (27Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38453) Google Scholar).TABLE 1Statistics on diffraction data and structure refinementData collectionPDE5A1 nativePDE5A1-sildenafilPDE5A1-icarisid IISpace groupP3121P6222P6122Unit cell (a, b, and c, Å)74.7, 74.7, 130.7164.6, 164.6, 193.1110.7, 110.7, 106.2Resolution (Å)1.852.31.8Total measurements363,9601,121,219412,999Unique reflections36,60368,78636,177Completeness (%)99.5 (100.0)aThe numbers in parentheses are for the highest resolution shell.99.9 (100.0)99.9 (99.7)Average I/σ20.8 (7.4)aThe numbers in parentheses are for the highest resolution shell.6.7 (4.8)13.6 (4.3)Rmerge0.058 (0.49)aThe numbers in parentheses are for the highest resolution shell.0.117 (0.58)0.061 (0.46)Structure refinementR-factor0.2210.2100.206R-free0.236 (9.7%)bThe percentage of reflections omitted for calculation of R-free.0.246 (9.7%)0.233 (9.7%)Resolution (Å)15-1.8548-2.344-1.8Reflections34,99666,31535,094r.m.s. deviationBond0.0078 Å0.00610.0073Angle1.25°1.17°1.31°Average B-factor (Å2)Protein37.5 (2523)cThe number of atoms in the crystallographic asymmetric unit.27.8 (7804)27.9 (2650)Inhibitor29.4 (99)25.9 (37)Water37.9 (148)cThe number of atoms in the crystallographic asymmetric unit.28.8 (311)32.8 (231)Zinc46.2 (1)cThe number of atoms in the crystallographic asymmetric unit.44.9 (3)36.6 (1)Magnesium33.4 (1)cThe number of atoms in the crystallographic asymmetric unit.37.6 (3)41.0 (1)a The numbers in parentheses are for the highest resolution shell.b The percentage of reflections omitted for calculation of R-free.c The number of atoms in the crystallographic asymmetric unit. Open table in a new tab Structure Determination—The structure of the unliganded PDE5A1 was solved by rigid body refinement of the PDE5A1 catalytic domain in PDE5A1-IBMX. The structures of PDE5A1 in complex with sildenafil and icarisid II were solved by the molecular replacement program AMoRe (28Navaza J. Saludjian P. Methods Enzymol. 1997; 276: 581-594Crossref PubMed Scopus (368) Google Scholar), using the PDE5A1-IBMX structure without the H-loop and IBMX as the initial model. The rotation and translation searches for the crystal of PDE5A1-icarisid II yielded a correlation coefficient of 0.74 and R-factor of 0.31 for 3054 reflections between 4 and 8 Å resolution. The rotation and translation searches for PDE5A1-sildenafil yielded a correlation coefficient of 0.22 and R-factor of 0.52 for 11,612 reflections between 4 and 8 Å resolution for the first molecule, and of 0.39 and 0.41 after the second molecule was added. The electron density map was improved by the density modification package of CCP4 (29Collaborative Computational Project, Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19734) Google Scholar). The atomic model was rebuilt by program O (30Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar) and refined by program CNS (Table 1) (31Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16949) Google Scholar). Multiple Conformations of the H-loop of PDE5—The crystallographic asymmetric units contain one molecule of the catalytic domain in the structures of the unliganded PDE5A1 and the icarisid II complex but three molecules in PDE5A1-sildenafil structure. The electron density maps showed that the entire catalytic domain in the PDE5A1-icarisid II structure and molecule A in the PDE5A1-sildenafil structure were traceable. However, residues 668-676 of molecules B and C in the PDE5A1-sildenafil crystal and residues 793-807 in the unliganded PDE5A1 lacked electron density and were disordered. The Ramachandran plots showed that the backbone conformations of 90-94% residues in the three structures were located in the most favored regions, and no residues were located in the energetically disallowed regions. The structures of the unliganded PDE5A1 catalytic domain (residues 535-860) and its complexes with sildenafil or icarisid II are composed of 14 common α-helices and a variable H-loop at the active site (Fig. 2). Structural superposition of the unliganded PDE5A1 over the complexes of PDE5A1-IBMX, PDE5A1-sildenafil, and PDE5A1-icarisid II yielded r.m.s. deviations of 0.29, 0.42, and 0.54 Å for the Cα atoms of comparable residues (536-659, 684-787, and 810-860), respectively, suggesting overall structural similarity. However, the H-loop (residues 660-683 on the basis of the structural comparison with seven other PDE families, which are slightly different from the previous assignment of residues 661-676 (23Huai Q. Liu Y. Francis S.H. Corbin J.D. Ke H. J. Biol. Chem. 2004; 279: 13095-13101Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar)) adopts four conformations and different tertiary structures in the crystals of the unliganded PDE5A1 and its complexes with IBMX, sildenafil, or icarisid II. In the unliganded PDE5A1 structure, the H-loop contains a few turns, but the majority of the H-loop residues are in a coil conformation (Fig. 2). Binding of IBMX converts the H-loop into two short α-helices involving residues 664-667 and 672-676 (Fig. 2F) and shifts the Cα atoms of the H-loop as much as 7 Å from those in the unliganded structure. In the structure of the PDE5-sildenafil complex, the H-loop in molecule A is converted to a turn and a 310 helix at residues 672-675, and the whole loop migrates as much as 24 Å to cover the active site (Fig. 2). However, residues 668-676 of the H-loop in molecules B and C are disordered. The most dramatic change in the H-loop occurs in the structure of PDE5A1-icarisid II, in which the H-loop contains two β-strands at residues 662-666 and 675-679 (Fig. 2F) and migrates as much as 35 Å from the position in the unliganded PDE5A1. To verify that the conformational changes are not because of an artifact of structure determination or crystal packing, electron density maps were calculated, and lattice interactions in the various crystal forms were examined. The maps that were calculated from the structure with omission of the H-loop showed solid electron density for almost all residues of the H-loops, thus confirming the true conformational variation in the PDE5A1 structures. This is supported by the fact that the B-factor for the H-loops is comparable with or slightly higher than the overall average B-factor for the protein atoms as follows: 48 versus 38 Å2 for the unliganded PDE5A1, 61 versus 40 Å2 for PDE5A1-IBMX, 33 versus 33 Å2 for PDE5A1-icarisid II, and 38 versus 27 Å2 for PDE5A1-sildenafil. In addition, the following facts suggest minor roles of the lattice contacts in the conformational changes of the H-loop. First, the unliganded PDE5A1 and its IBMX complex have the same space group and the similar unit cell parameters (a = b = 74.7, c = 130.7 Å versus a = b = 74.5, c = 130.1 Å) but different H-loop conformations. Second, the PDE4 H-loop that was inserted into the chimeric PDE5 structure is involved in the crystal lattice interactions of PDE5 but retains its conformation found in PDE4 (22Zhang K.Y. Card G.L. Suzuki Y. Artis D.R. Fong D. Gillette S. Hsieh D. Neiman J. West B.L. Zhang C. Milburn M.V. Kim S.H. Schlessinger J. Bollag G. Mol. Cell. 2004; 15: 279-2861Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Thus, the dramatic conformational changes of the H-loop must be the consequence of binding of the specific inhibitors. In addition to variation of the H-loop, minor conformational differences are observed for another active site loop, the M-loop (residues 788-811 on the basis of the structural comparison among seven PDE families, in comparison to the original assignment of residues 787-812 (23Huai Q. Liu Y. Francis S.H. Corbin J.D. Ke H. J. Biol. Chem. 2004; 279: 13095-13101Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar)). Residues 793-807 of the M-loop are not traceable in the structures of the unliganded or IBMX-bound PDE5A1. However, the well ordered M-loops in the structures of PDE5A1 in complex with sildenafil or icarisid II contain an extra 310 helix and a 10-residue extension of α-helix H14, in addition to the correspondence of a 310 helix to the N-terminal portion of H15 in the unliganded PDE5A1 (Fig. 2F). PDE5 shows an apparently unique feature distinct from other PDE families, although the overall topological folding of PDE5A is similar to those of PDE1B (22Zhang K.Y. Card G.L. Suzuki Y. Artis D.R. Fong D. Gillette S. Hsieh D. Neiman J. West B.L. Zhang C. Milburn M.V. Kim S.H. Schlessinger J. Bollag G. Mol. Cell. 2004; 15: 279-2861Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), PDE2A (32Iffland A. Kohls D. Low S. Luan J. Zhang Y. Kothe M. Cao Q. Kamath A.V. Ding Y.H. Ellenberger T. Biochemistry. 2005; 44: 8312-8325Crossref PubMed Scopus (55) Google Scholar), PDE3B (33Scapin G. Patel S.B. Chung C. Varnerin J.P. Edmondson S.D. Mastracchio A. Parmee E.R. Singh S.B. Becker J.W. Van der Ploeg L.H. Tota M.R. Biochemistry. 2004; 43: 6091-6100Crossref PubMed Scopus (97) Google Scholar), PDE4B and PDE4D (22Zhang K.Y. Card G.L. Suzuki Y. Artis D.R. Fong D. Gillette S. Hsieh D. Neiman J. West B.L. Zhang C. Milburn M.V. Kim S.H. Schlessinger J. Bollag G. Mol. Cell. 2004; 15: 279-2861Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 23Huai Q. Liu Y. Francis S.H. Corbin J.D. Ke H. J. Biol. Chem. 2004; 279: 13095-13101Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 24Card G.L. England B.P. Suzuki Y. Fong D. Powell B. Lee B. Luu C. Tabrizizad M. Gillette S. Ibrahim P.N. Artis D.R. Bollag G. Milburn M.V. Kim S.H. Schlessinger J. Zhang K.Y. Structure (Camb.). 2004; 12: 2233-2247Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 34Xu R.X. Hassell A.M. Vanderwall D. Lambert M.H. Holmes W.D. Luther M.A. Rocque W.J. Milburn M.V. Zhao Y. Ke H. Nolte R.T. Science. 2000; 288: 1822-1825Crossref PubMed Scopus (318) Google Scholar, 35Xu R.X. Rocque W.J. Lambert M.H. Vanderwall D.E. Luther M.A. Nolte R.T. J. Mol. Biol. 2004; 337: 355-365Crossref PubMed Scopus (108) Google Scholar, 36Lee M.E. Markowitz J. Lee J.O. Lee H. FEBS Lett. 2002; 530: 53-58Crossref PubMed Scopus (123) Google Scholar, 37Huai Q. Wang H. Sun Y. Kim H.Y. Liu Y. Ke H. Structure (Camb.). 2003; 11: 865-873Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 38Huai Q. Colicelli J. Ke H. Biochemistry. 2003; 42: 13220-13226Crossref PubMed Scopus (110) Google Scholar), PDE7A (25Wang H. Liu Y. Chen Y. Robinson H. Ke H. J. Biol. Chem. 2005; 280: 30949-30955Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), and PDE9A (39Huai Q. Wang H. Zhang W. Colman R. Robinson H. Ke H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9624-9629Crossref PubMed Scopus (87) Google Scholar). The core catalytic domains of PDE1-4, -7, and -9 (residues 115-411 in PDE4D2), including the H-loop that is composed of two short α-helices (H8 and H9, residues 209-215 and 218-222 in PDE4D2l; see Fig. 2F) have a uniform conformation and are superimposable on one another. In contrast, the H-loop of PDE5A1 presents at least four different conformations, and none of these is comparable with any of the corresponding H-loops in other PDE families. The closest comparable conformation is the H-loop in the PDE5A1-IBMX structure, which also contains two short α-helices. However, these two helices have as much as 7 Å positional difference from those in PDE4D2 (23Huai Q. Liu Y. Francis S.H. Corbin J.D. Ke H. J. Biol. Chem. 2004; 279: 13095-13101Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and are in a different three-dimensional arrangement. In addition, the β-strand component of the H-loop in the PDE5A1-icarisid II complex is unique among active sites of known PDE structures. Therefore, the active site of PDE5 appears to belong to a special category of the PDE superfamily. Conformation Variation of Sildenafil—Sildenafil binds to each active site of three PDE5A1 catalytic domains in the crystallographic asymmetric unit with similar conformation and occupancy, as shown by the comparable B-factor and the clean electron density in the omitted map (Fig. 3). The binding of sildenafil causes a dramatic conformational change of the H-loop and a movement as much as 24 Å from that in the unliganded PDE5 structure. A direct consequence of the H-loop movement is the transformation of the open PDE5A1 active site to a closed pocket. Sildenafil is partially buried in the pocket. Solvent-accessible surface of sildenafil after binding to PDE5A1 is reduced to 9.4% of the total surface area. Sildenafil borders the metalbinding pocket but does not directly interact with the metal ions. The pyrazolopyrimidinone group (R1 in Fig. 1 and Table 2) of sildenafil stacks against Phe820 of PDE5A1 and also contacts residues Tyr612, Leu765, Ala767, and Gln817. The O1 and N4 atoms of pyrazolopyrimidinone form two hydrogen bonds with Nϵ2 and Oϵ1ofGln817, respectively. The ethoxyphenyl group (R2, Fig. 1) interacts via van der Waals forces with Val782, Ala783, Phe786, Leu804, Ile813, Gln817, and Phe820. The methylpiperazine group (R, Fig. 1) contacts Asn662, Ser6633, Tyr664, Ile665, Leu804, and Phe820. The oxygen atoms of the sulfone group interact mainly with Phe820.TABLE 2Interactions of sildenafil and icarisid II with PDE5AView Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table