Phosphodiesterase-5 Gln817 Is Critical for cGMP, Vardenafil, or Sildenafil Affinity
2006; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m510372200
ISSN1083-351X
AutoresRoya Zoraghi, Jackie D. Corbin, Sharron H. Francis,
Tópico(s)Chemical synthesis and alkaloids
ResumoThe side group of an invariant Gln in cGMP- and cAMP-specific phosphodiesterases (PDE) is held in different orientations by bonds with other amino acids and purportedly discriminates between guanine and adenine in cGMP and cAMP. In cGMP-specific PDE5, Gln775 constrains the orientation of the invariant Gln817 side chain, which forms bidentate bonds with 5′-GMP, vardenafil, sildenafil, and 3-isobutyl-1-methylxanthine (IBMX) (Sung, 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., and Cho, J. M. (2003) Nature 425, 98–102; Huai, Q., Liu, Y., Francis, S. H., Corbin, J. D., and Ke, H. (2004) J. Biol. Chem. 279, 13095–13101; Zhang, 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., and Bollag, G. (2004) Mol. Cell 15, 279–286). PDE5Q817A and PDE5Q775A were generated to test the hypotheses that Gln817 is critical for cyclic nucleotide or inhibitor affinity and that Gln775 immobilizes the Gln817 side chain to provide cGMP/cAMP selectivity. Allosteric cGMP binding and the molecular mass of the mutant proteins were unchanged compared with PDE5WT. For PDE5Q817A, Km for cGMP or cAMP was weakened 60- or 2-fold, respectively. For PDE5Q775A, Km for cGMP was weakened ∼20-fold but was unchanged for cAMP. For PDE5Q817A, vardenafil, sildenafil, and IBMX inhibitory potencies were weakened 610-, 48-, and 60-fold, respectively, indicating that Gln817 is a major determinant of potency, especially for vardenafil, and that binding of vardenafil and sildenafil differs substantially. Sildenafil and vardenafil affinity were not significantly affected in PDE5Q775A. It is concluded that Gln817 is a positive determinant for PDE5 affinity for cGMP and several inhibitors; Gln775, which perhaps restricts rotation of Gln817 side chain, is critical for cGMP affinity but has no measurable effect on affinity for cAMP, sildenafil, or vardenafil. The side group of an invariant Gln in cGMP- and cAMP-specific phosphodiesterases (PDE) is held in different orientations by bonds with other amino acids and purportedly discriminates between guanine and adenine in cGMP and cAMP. In cGMP-specific PDE5, Gln775 constrains the orientation of the invariant Gln817 side chain, which forms bidentate bonds with 5′-GMP, vardenafil, sildenafil, and 3-isobutyl-1-methylxanthine (IBMX) (Sung, 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., and Cho, J. M. (2003) Nature 425, 98–102; Huai, Q., Liu, Y., Francis, S. H., Corbin, J. D., and Ke, H. (2004) J. Biol. Chem. 279, 13095–13101; Zhang, 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., and Bollag, G. (2004) Mol. Cell 15, 279–286). PDE5Q817A and PDE5Q775A were generated to test the hypotheses that Gln817 is critical for cyclic nucleotide or inhibitor affinity and that Gln775 immobilizes the Gln817 side chain to provide cGMP/cAMP selectivity. Allosteric cGMP binding and the molecular mass of the mutant proteins were unchanged compared with PDE5WT. For PDE5Q817A, Km for cGMP or cAMP was weakened 60- or 2-fold, respectively. For PDE5Q775A, Km for cGMP was weakened ∼20-fold but was unchanged for cAMP. For PDE5Q817A, vardenafil, sildenafil, and IBMX inhibitory potencies were weakened 610-, 48-, and 60-fold, respectively, indicating that Gln817 is a major determinant of potency, especially for vardenafil, and that binding of vardenafil and sildenafil differs substantially. Sildenafil and vardenafil affinity were not significantly affected in PDE5Q775A. It is concluded that Gln817 is a positive determinant for PDE5 affinity for cGMP and several inhibitors; Gln775, which perhaps restricts rotation of Gln817 side chain, is critical for cGMP affinity but has no measurable effect on affinity for cAMP, sildenafil, or vardenafil. Cyclic nucleotide (cN) 2The abbreviations used are: cN, cyclic nucleotide(s); PDE, cyclic nucleotide phosphodiesterase; IBMX, 3-isobutyl-1-methylxanthine; C domain, catalytic domain. 2The abbreviations used are: cN, cyclic nucleotide(s); PDE, cyclic nucleotide phosphodiesterase; IBMX, 3-isobutyl-1-methylxanthine; C domain, catalytic domain. phosphodiesterases (PDE) cleave the 3′,5′-cyclic phosphate bond of cNs. Among cGMP- or cAMP-specific PDEs, affinities for the cNs differ by 100-fold or more (1Houslay M.D. Adams D.R. Biochem. J. 2003; 370: 1-18Crossref PubMed Scopus (642) Google Scholar, 2Beavo J.A. Physiol. Rev. 1995; 75: 725-748Crossref PubMed Scopus (1626) Google Scholar, 3Soderling S.H. Beavo J.A. Curr. Opin. Cell Biol. 2000; 12: 174-179Crossref PubMed Scopus (641) Google Scholar, 4Francis S.H. Turko I.V. Corbin J.D. Prog. Nucleic Acid Res. Mol. Biol. 2001; 65: 1-52Crossref PubMed Google Scholar). Co-crystals of PDE catalytic domains (C domain) with the products provide insight into mechanisms contributing to cAMP and cGMP binding and selectivity (5Lee M.E. Markowitz J. Lee J.O. Lee H. FEBS Lett. 2002; 530: 53-58Crossref PubMed Scopus (123) Google Scholar, 6Huai 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, 7Huai Q. Colicelli J. Ke H. Biochemistry. 2003; 42: 13220-13226Crossref PubMed Scopus (109) Google Scholar, 8Xu 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 (107) Google Scholar, 9Scapin 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, 10Huai Q. Wang H. Zhang W. Colman R.W. Robinson H. Ke H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9624-9629Crossref PubMed Scopus (86) Google Scholar, 11Conti M. Nat. Struct. Mol. Biol. 2004; 11: 809-810Crossref PubMed Scopus (16) Google Scholar). In addition, inhibitors and 5′-GMP form some of the same contacts in PDE5 (12Sung 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 (231) Google Scholar, 13Huai 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 (114) Google Scholar, 14Zhang 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: 279-286Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Information from the crystal structure of the cAMP-specific PDE4B led Xu et al. (15Xu 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 (317) Google Scholar) to propose that an invariant glutamine might provide for cN selectivity (Fig. 1A); they noted that the side chain of the invariant glutamine (Gln443) is held in place by hydrogen bond (H-bond) with the Tyr403 hydroxyl and in this position forms a bidentate H-bond with two positions in adenine of cAMP (Fig. 1A). Xu et al. (15Xu 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 (317) Google Scholar) suggested that the side chain of the invariant glutamine could rotate ∼180° so that the positions of the O∈ and N∈ of the side chain would be reversed and could form a bidentate H-bond complex with the guanine of cGMP, which has different H-bonding potential. As predicted, in crystal structures of cGMP-specific PDE5, the invariant glutamine (Gln817) side chain is immobilized by H-bond to Gln775 (12Sung 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 (231) Google Scholar, 13Huai 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 (114) Google Scholar). In a co-crystal structure of PDE5 C domain with the 5′-GMP product, the Gln817 side chain forms a bidentate H-bond with the N-1 proton and C-6 carbonyl oxygen of guanine (Fig. 1A) (14Zhang 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: 279-286Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). In x-ray crystal structures of dual specificity PDEs, the side chain of the invariant glutamine can adopt both configurations shown in Fig. 1A, allowing for interaction with either cN (9Scapin 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, 14Zhang 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: 279-286Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Based on these observations, orientation of the side chain of the invariant glutamine was referred to as the "glutamine switch" (14Zhang 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: 279-286Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). Crystal structures of PDE5 C domain bound with the inhibitors sildenafil or vardenafil revealed that atoms in these inhibitors that are analogous to substituents at N-1 and C-6 in cGMP form a bidentate H-bond with the Gln817 side chain (Fig. 1B) (12Sung 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 (231) Google Scholar). 3-Isobutyl-1-methylxanthine (IBMX), a weak inhibitor, also forms a bidentate H-bond with Gln817, but its orientation and interactions differ (Fig. 1B) (13Huai 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 (114) Google Scholar). X-ray crystal structures of isolated C domains of PDEs co-crystallized with ligands provide invaluable insights into contacts and topography of the catalytic sites. However, the importance of these contacts must be experimentally quantified in holoenzymes to assess the role of these interactions in the enzyme-ligand complex and to provide appropriate direction for improved drug design. The combined approaches provide a more accurate profile of catalytic site function. Based on the prediction of the glutamine switch as the determinant of cN selectivity in PDEs and supporting x-ray crystallographic evidence, we hypothesized that Gln817 is a critical contact for interaction with cGMP, vardenafil, sildenafil, or IBMX and that H-bonding of the Gln817 side chain with Gln775 optimizes the configuration for contact with cGMP, vardenafil, sildenafil, and IBMX but not with cAMP. Removal of the H-bonding potential provided by Gln775 was predicted to free the rotational constraint of Gln817, weaken affinity for cGMP, and improve affinity for cAMP substantially. To address these questions, point mutations of Gln817 and Gln775 were created to determine (a) the importance of Gln817 in providing for potency of interaction of PDE5 with cGMP, cAMP, or inhibitors and (b) the role of Gln775 in providing for cGMP versus cAMP specificity in PDE5 and its impact on sildenafil or vardenafil potency. Materials—[3H]cGMP was purchased from Amersham Biosciences. IBMX, Crotalus atrox snake venom, cGMP, and histone type II-AS were obtained from Sigma. Sildenafil was purified from Viagra tablets as described in our earlier report (16Francis S.H. Sekhar K.R. Rouse A.B. Grimes K.A. Corbin J.D. Int. J. Impot. Res. 2003; 15: 369-372Crossref PubMed Scopus (23) Google Scholar). Generation of Wild-type and Mutant (Q817A and Q775A) hPDE5A1— Human cDNA coding for full-length PDE5A1 (courtesy of Dr. K. Omori, Tanabe-Seiyaku Pharmaceutical Co. Ltd., Saitama, Japan) was used as the template to generate full-length PDE5A1 by introduction of start and stop codons at appropriate loci. The resulting PCR fragment was cloned into pCR 2.1-TOPO® vector (Invitrogen), verified by sequencing, and then ligated into the EcoRI and NotI unique sites of the baculovirus expression vector pAcHLT-A (Pharmingen). pAcHLT-A vector contains a His6 sequence that precedes the coding region. This step resulted in plasmid pAcA-PDE5 (Met1–Asn875), which generated N-terminally His-tagged recombinant hPDE5A1. The QuikChange site-directed mutagenesis kit (Stratagene) was used to make point mutations (Gln817 to Ala (Q817A) and Gln775 to Ala (Q775A)) in the pAcA-PDE5 expression vector according to the manufacturer's protocol using the following pairs of mutagenic oligonucleotides (altered bases are underlined): 1) for Q817A, 5′-ATCCCAAGTATGGCAGTTGGGTTCATAG-3′ and 5′-CTATGAACCCAACTGCCATACTTGGGAT-3′; 2) for Q775A, 5′-AACCCTGGCCTATTGCACAACGGATAGCAG-3′ and 5′-CTGCTATCCGTTGTGCAATAGGCCAGGGTT-3′. The presence of the desired mutation was verified by sequencing the entire DNA segment. Expression of Wild-type and Mutant hPDE5A1—Sf9 cells (BD Pharmingen) were cotransfected with BaculoGold linear baculovirus DNA (BD Pharmingen) and one of the hPDE5A1 constructs (PDE5WT (Met1–Asn875), PDE5Q817A (Met1–Asn875)Q817A, and PDE5Q775A (Met1–Asn875)Q775A) in the pAcHLT-A baculovirus expression vector by the calcium phosphate method according to the protocol from BD Pharmingen. At 5 days post-infection, the cotransfection supernatant was collected, amplified three times in Sf9 cells, and used directly as viral stock for expression without additional purification of recombinant viruses. Sf9 cells grown at 27 °C in complete Grace's insect medium with 10% fetal bovine serum and 10 μg/ml gentamicin (Sigma) in T-175 flasks (Corning) were typically infected with 100 μl of viral stock/flask and harvested 92 h post-infection. Purification of Wild-type and Mutant hPDE5A1—Purification steps were done at 4 °C. The Sf9 cell pellet for each T-175 flask (∼2 × 107 cells) was resuspended in 3 ml of ice-cold lysis buffer (20 mm Tris-HCl, pH 8, 100 mm NaCl) containing Complete™ protease inhibitor (Roche Molecular Biochemicals) as recommended by the manufacturer. Cell suspension was homogenized in 10–20-ml aliquots on ice by two 6-s bursts in an Ultra Turrex microhomogenizer (Tekmar) with a 20-s recovery between bursts. Cell homogenate was centrifuged (20 min, 10,000 rpm, in a Beckman JA-20 rotor). The supernatant was applied to a nickel-nitrilotriacetic acid-agarose (Qiagen) column (1 × 2 cm) equilibrated with lysis buffer. The column was sequentially washed with 100 ml of lysis buffer and a stepwise gradient of imidazole (0.8 to 20 mm) in lysis buffer. Lysis buffer containing 100 mm imidazole was soaked into the resin, and after 2 h, ten 1-ml fractions were collected. Elutions containing PDE5 protein were dialyzed versus 2000 volumes of 10 mm potassium phosphate, pH 6.8, 25 mm β-mercaptoethanol, and 150 mm NaCl, flash-frozen in the same buffer containing 10% sucrose, and stored at –70 °C. Activity in frozen samples was stable for at least 10 months. SDS-PAGE of hPDE5A1 Constructs—The purity and integrity of proteins were assessed using SDS-PAGE. Protein samples were boiled for 4 min in the presence of 10% SDS, 2 m β-mercaptoethanol, and 0.1% bromphenol blue and subjected to 12% SDS-polyacrylamide gel electrophoresis before visualization by Coomassie Brilliant Blue staining. cGMP Binding—To measure allosteric cGMP-binding, Millipore filter binding assays were conducted in a total volume of 50 μl of reaction mixture that contained 10 mm potassium phosphate buffer, pH 6.8, 1 mm EDTA, 0.5 mg/ml histone type II-AS, 30 mm dl-dithiothreitol, 0.2 mm sildenafil, and either 3 μm (for stoichoimetry determination) or 0.05–4 μm (for binding affinity determination) [3H]cGMP. The binding reaction was initiated by the addition of enzyme and incubated at 4 °C for 60 min. 1 ml of cold KP buffer (10 mm potassium phosphate, pH 6.8) was added to each sample, and samples were filtered immediately onto premoistened Millipore HAWP filters (pore size 0.45 μm). Filters were washed twice with 2 ml of cold KP buffer, dried, and counted using non-aqueous Ready Safe scintillation mixture (Beckman). Counts bound to PDE5 were corrected by subtraction of nonspecific binding (+1 mm unlabeled cGMP). Blanks containing no PDE5 were run for each [3H]cGMP concentration. Catalytic Activity—PDE activity was determined as described (17Francis S.H. Lincoln T.M. Corbin J.D. J. Biol. Chem. 1980; 255: 620-626Abstract Full Text PDF PubMed Google Scholar). Reaction mixture contained 50 mm Tris HCl, pH 7.5, 10 mm MgCl2, 0.3 mg/ml bovine serum albumin, and either cGMP (0–750 μm and [3H]cGMP (60,000–150,000 cpm/assay tube)) or cAMP (0–1500 μm [3H]cAMP (60,000–150,000 cpm/assay tube)) as substrate and one of the PDE5 proteins in a total volume of 50–100 μl. Incubation time was 10–20 min at 30 °C. Apparent Km and Vmax were determined by nonlinear regression analysis of data using Prism Graphpad software. In all studies, <10% of total [3H]cNMP was hydrolyzed. To determine IC50 for sildenafil, vardenafil, or IBMX, PDE catalytic activity was assayed in triplicate in the presence of a range of inhibitor concentrations (1–1,000,000,000 pm) with 0.5 μm cGMP as substrate. Ki values were calculated using the equation Ki = IC50/1 + [S]/Km. Calculation of Free Energy of Binding—The Gibbs free energy change, ΔG, which occurs by association of a ligand with PDE5, is related to the equilibrium association constant for the interaction and was calculated using Equation 1,ΔG=-RTlnK(Eq. 1) where K = Km or Ki calculated from IC50 values for the inhibitors as described above R is the ideal gas constant (equal to 1.98 × 10–3 kcal/degree/mol), and T is the temperature at which the assay was done (303 K). Values reported represent three measurements, each in triplicate. High-affinity interaction is indicated when ΔG is a large negative value. The contribution of a substituted amino acid side chain to the Gibbs free energy of binding in enzyme-transition state complexes was calculated from the biochemical potency (Km or Ki values calculated from the IC50 values for the inhibitors) using Equation 2,ΔΔG=ΔGWT-ΔGmut(Eq. 2) where ΔΔG is the change in free energy of binding in enzyme-transition state complexes attributable to the substituted group (18Wilkinson A.J. Fersht A.R. Blow D.M. Winter G. Biochemistry. 1983; 22: 3581-3586Crossref PubMed Scopus (251) Google Scholar, 19Andrews P.R. Craik D.J. Martin J.L. J. Med. Chem. 1984; 27: 1648-1657Crossref PubMed Scopus (452) Google Scholar). Enzymes in these studies were His-tagged constructs that had been expressed in Sf9 cells, purified, and characterized as described under "Experimental Procedures." Structural integrity of each was verified by pattern of migration on SDS-PAGE and allosteric cGMP-binding properties (see supplemental material). Effect of Gln817 Substitution on PDE5 Catalytic Function—Kinetic characteristics of catalytic function of purified PDE5 constructs were determined as described under "Experimental Procedures" (Fig. 2, Table 1). PDE5WT had a Km for cGMP of 2.9 ± 0.8 μm and a kcat of 2.2 ± 0.4 s–1.Gln817 was replaced by alanine (PDE5Q817A), which lacks the O∈ and N∈ groups that in the PDE5 crystal structure were shown to form a bidentate H-bond with 5′-GMP, sildenafil, or vardenafil; these contacts were inferred to also occur with cGMP. PDE5Q817A catalytic site affinity for cGMP was decreased 60-fold (Km = 180 ± 5.2 μm) (Fig. 2A, Table 1), indicating that the Gln817 side chain is extremely important for high-affinity interaction with cGMP; kcat (3.8 ± 0.2 s–1) was slightly improved.TABLE 1Effect of PDE5 Q817A and Q775A mutations on affinity for cGMP and cAMP as substrateshPDE5A1 proteinscGMP KmcGMP KmcGMP KcatcGMP Kcat/KmcAMP KmcAMP KmcAMP KcatcAMP Kcat/KmcN selectivityμm-fold effects-1×106 mol-1 s-1μm-fold effects-1×106 mol-1 s-1KmcAMP/KmcGMPWT2.9 ± 0.812.2 ± 0.40.758290 ± 8.011.6 ± 0.20.005100Q817A180 ± 5.2603.8 ± 0.20.021630 ± 4223.0 ± 0.30.0053.5Q775A60 ± 3.3204.6 ± 0.20.076247 ± 1813.4 ± 0.20.0144.1 Open table in a new tab PDE5WT hydrolyzes cAMP with ∼100-fold lower affinity (Km = 290 ± 8.0 μm) than for cGMP; kcat for cAMP hydrolysis was 1.6 ± 0.2 s–1. PDE5Q817A affinity for cAMP (Km = 630 ± 42 μm) was slightly weaker (∼2-fold) than that of PDE5WT; kcat (3.0 ± 0.3 s–1) was slightly improved (Fig. 2B, Table 1). Catalytic efficiency (kcat/Km) for hydrolysis of cGMP declined 36-fold in PDEQ817A (Table 1) but was unchanged for cAMP. The selective impact of removal of the Gln817 side chain on affinity and catalytic efficiency for cGMP versus cAMP as substrate was consistent with the importance of this contact with cGMP, which was inferred from the co-crystal of PDE5 and 5′-GMP. The lack of a deleterious effect of the mutation on kcat suggested that the mutation did not introduce a globally deleterious effect on the catalytic site. Effect of Gln775 Substitution on PDE5 Catalytic Function—The kcat (4.6 ± 0.2 s–1) for hydrolysis of cGMP and kcat (3.4 ± 0.2) for hydrolysis of cAMP by PDE5Q775A were slightly improved compared with PDE5WT (Table 1). Km for cGMP was weakened 20-fold (60 ± 3.3 μm) compared with that for PDE5WT (Fig. 2A, Table 1), but Km for cAMP was essentially unchanged (Km = 247 ± 18 μm) (Fig. 2B, Table 1). Catalytic efficiency (kcat/Km) of PDE5Q775A for cGMP was decreased 10-fold and improved ∼3-fold for cAMP. Selectivity for cGMP over cAMP declined from ∼100-fold in PDE5WT to ∼4-fold in both mutants, but this was due to the loss in affinity for cGMP. These results indicated that restriction of the orientation of the Gln817 side arm by Gln775 is a positive determinant for cGMP affinity in the PDE5 catalytic site, but removal of that restriction does not improve cAMP affinity. This brings into question the importance of the orientation of the side chain of the invariant glutamine in determining cN selectivity in other PDEs as well. In the crystal structure of the PDE9 C domain, a cGMP-specific PDE, the side chain of the invariant glutamine is not tethered to restrict orientation (10Huai Q. Wang H. Zhang W. Colman R.W. Robinson H. Ke H. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9624-9629Crossref PubMed Scopus (86) Google Scholar). In combination, the results provided herein refute the assertion that an H-bond constraint of the glutamine side chain in cGMP-specific PDEs provides for discrimination against cAMP. In addition, the 20-fold change in affinity of PDEQ775A for cGMP suggests that Gln775 may contribute to cGMP binding affinity by processes not mediated through Gln817. Effect of Gln817 Substitution on Affinity of PDE5 Catalytic Site for Inhibitors—X-ray co-crystal structures of PDE5 with vardenafil, sildenafil, or IBMX revealed that, like 5′-GMP, all three inhibitors form contacts with the Gln817 side chain (12Sung 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 (231) Google Scholar, 13Huai 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 (114) Google Scholar). To quantify the role of the Gln817 side chain for inhibitor potencies, IC50 values were determined as described under "Experimental Procedures." Substrate concentration (0.5 μm [3H]cGMP) was significantly lower than the Km for each construct, so that IC50 should approach Ki. Potency of inhibition of PDE5Q817A by vardenafil, sildenafil, or IBMX (IC50 values of 61, 141, or 214,000 nm, respectively) was substantially weaker than that for PDE5WT (IC50 values of 0.1, 2.9, and 3500 nm, respectively) (Fig. 3, Table 2). The dramatic difference in effect of the Q817A mutation on affinity for vardenafil (610-fold) versus cGMP (60-fold) or sildenafil (48-fold) indicated that despite purportedly similar interactions between Gln817 and these ligands, as indicated in the x-ray crystal structures, significant quantitative differences exist. The 13-fold disparity between the effect on potencies of vardenafil and sildenafil challenges the interpretation that the two inhibitors bind similarly at this position in PDE5. The relative selectivity for vardenafil over sildenafil in PDE5Q817A declined sharply from 29-fold in PDE5wt to 2.3-fold in PDE5Q817A (Table 2). This emphasizes the particular importance of Gln817 as a determinant of vardenafil potency and selectivity between vardenafil and sildenafil. The similar change in potency for sildenafil and IBMX (48- and 60-fold, respectively) was surprising and suggests that despite the difference in potency (∼1200-fold) of these inhibitors and the H-bonding pattern (Fig. 1B), the relative importance of Gln817 for binding of each inhibitor is similar.TABLE 2Comparison of the effect of PDE5 Q817A mutation on potency of inhibition by vardenafil, sildenafil, or IBMXhPDE5A1IC50IC50 -fold effectIC50vardenafil/ IC50sildenafil selectivityVardenafilSildenafilIBMXVardenafilSildenafilIBMXnmnmμmnmnmμm-foldWild type0.1 ± 0.022.9 ± 0.43.5 ± 0.311129Q817A61 ± 3.1141 ± 3.5214 ± 4.361048602.3Q775A0.08 ± 0.012.6 ± 0.4ND0.80.9ND33 Open table in a new tab Effect of Gln775 Substitution on PDE5 Affinity for Sildenafil or Vardenafil—The potency of sildenafil or vardenafil inhibition of PDEQ775A catalytic activity, assessed as described under "Experimental Procedures," did not differ significantly from that of PDE5WT (Table 2). Thus, freeing the putative rotation potential of the Gln817 side chain had little if any effect on the affinity for these inhibitors compared with a 20-fold effect on affinity for cGMP. Contribution of Gln817 to Free Energy of Binding of cGMP and PDE Inhibitors—The interactions of cGMP, vardenafil, sildenafil, or IBMX with PDE5WT were quantified by calculating the Gibbs free energy of binding (ΔG) of 7.7, 13.8. 11.9, and 7.6 kcal mol–1, respectively, as described under "Experimental Procedures" (18Wilkinson A.J. Fersht A.R. Blow D.M. Winter G. Biochemistry. 1983; 22: 3581-3586Crossref PubMed Scopus (251) Google Scholar, 19Andrews P.R. Craik D.J. Martin J.L. J. Med. Chem. 1984; 27: 1648-1657Crossref PubMed Scopus (452) Google Scholar), and using the Km for cGMP or the Ki calculated from the respective IC50 values for the inhibitors. IC50 was determined under conditions in which IC50 should not differ substantially from the Ki. The change in free energy of binding (ΔΔG) of PDE5Q817A for cGMP, vardenafil, sildenafil, or IBMX (2.5, 3.8, 2.4, and 2.5 kcal mol–1) was consistent with loss of one H-bond because an H-bond contribution to the binding energy of a ligand for its receptor is 2.5–4 kcal mol–1 (18Wilkinson A.J. Fersht A.R. Blow D.M. Winter G. Biochemistry. 1983; 22: 3581-3586Crossref PubMed Scopus (251) Google Scholar, 19Andrews P.R. Craik D.J. Martin J.L. J. Med. Chem. 1984; 27: 1648-1657Crossref PubMed Scopus (452) Google Scholar). ΔΔG for loss in PDE5Q775A affinity for cGMP was 1.9 kcal mol–1. Concluding Remarks—Mutation of invariant Gln817 causes a ∼60-fold decline in cGMP affinity but only a slight change in cAMP affinity; the decline in cGMP/cAMP selectivity from 100-fold to ∼4-fold is due almost entirely to loss in cGMP binding affinity. As indicated by ablation of the H-bond provided by Gln775 to immobilize the Gln817 side chain, rotational constraint of the Gln817 side chain is a positive determinant for cGMP binding affinity but does not affect cAMP affinity. Thus, contrary to accepted dogma, features in the PDE5 catalytic site other than Gln817 and the orientation of its side group restrict PDE5 affinity for cAMP. This challenges the interpretation that rotational constraint of the glutamine switch is a major determinant of PDE cN selectivity. Discrimination between the two cNs in the various PDE catalytic sites is likely to be mechanistically diverse and to result from subtle differences in the various sites. Evidence suggests that the invariant glutamine can in some instances provide a substantial portion of the affinity for interaction with cGMP or cAMP. However, cN selectivity of a site relies on multiple factors including access of the cN to the pocket and formation of multiple contacts within the site. In solution, cGMP and cAMP are in equilibrium between syn and anti conformers. It seems unlikely that conformational features of the cNs in solution influence PDE catalytic site selectivity, but different catalytic sites could impose conformational changes on the cNs, thereby influencing contacts that foster high affinity. The 20-fold decline in affinity for cGMP upon removal of Gln775 (PDE5Q775A) could suggest that Gln775 directly contacts cGMP or stabilizes other important interactions. A co-crystal structure of PDE5 with cGMP will be required to resolve these possibilities. Notably, there was no change in affinity for sildenafil or vardenafil in PDE5Q775A. although the side chain of Gln817 would be predicted to be in an unfavorable orientation for binding the inhibitors part of the time. This reveals further differences in the binding of cGMP and sildenafil or vardenafil. The contribution of Gln817 to PDE5 affinity for vardenafil is 10-fold greater than that for cGMP or sildenafil. This could be due to a substantially stronger bidentate H-bond between the Gln817 side chain and vardenafil than with the other ligands, but electronegativity of the pyrimidine N-1 does not differ significantly between vardenafil and sildenafil (data not shown). Alternatively, Gln817 could influence other contacts between vardenafil and PDE5 more than for other ligands. Gln817 is also critical for PDE5 selectivity for vardenafil over sildenafil. The results presented herein and in our previous report (20Zoraghi R. Francis S.H. Wood D. Bischoff E. Hutter J. Knorr A. Niedbala M. Enyedy I. Pomerantz K. Corbin J.D. Int. J. Impot. Res. 2005; (in press)PubMed Google Scholar) emphasize that interpretation of the importance of contacts detected in x-ray co-crystal structures of PDE C domains and ligands is limited. The energetic contribution of each amino acid must be experimentally quantified in holoenzymes to more accurately assess the role of interactions and to provide direction for improved drug design. A comparison of differences in the effect of alanine substitution for Gln817 (60-fold) or Tyr612 (15-fold) (20Zoraghi R. Francis S.H. Wood D. Bischoff E. Hutter J. Knorr A. Niedbala M. Enyedy I. Pomerantz K. Corbin J.D. Int. J. Impot. Res. 2005; (in press)PubMed Google Scholar) on cGMP affinity or potency of inhibition by vardenafil, sildenafil, and IBMX reveals that Gln817 plays a central role in binding each of these. The unexpected differences in the role of Gln817 in the potency of vardenafil, sildenafil, and IBMX may be due in part to the fact that current studies used PDE5 holoenzyme, whereas the crystal structures utilized isolated C domain in complex with ligands. More consideration must be given to the impact of the regulatory domain of PDEs on ligand interaction at the catalytic site because holoenzymes are the pharmacological targets (21Corbin J.D. Turko I.V. Beasley A. Francis S.H. Eur. J. Biochem. 2000; 267: 2760-2767Crossref PubMed Scopus (224) Google Scholar, 22Mullershausen F. Friebe A. Feil R. Thompson W.J. Hofmann F. Koesling D. J. Cell Biol. 2003; 160: 719-727Crossref PubMed Scopus (137) Google Scholar, 23Rybalkin S.D. Rybalkina I.G. Shimizu-Albergine M. Tang X.B. Beavo J. EMBO J. 2003; 22: 469-478Crossref PubMed Scopus (193) Google Scholar, 24Blount M.A. Beasley A. Zoraghi R. Sekhar K.R. Bessay E.P. Francis S.H. Corbin J.D. Mol. Pharmacol. 2004; 66: 144-152Crossref PubMed Scopus (147) Google Scholar). Subtle changes in catalytic site topography may manifest as marked changes in function and affinity for ligands. Results from both x-ray crystallography and mutational analyses have limitations, but the combined approaches provide critical direction in rational design of highly specific and potent inhibitors for PDEs. We especially thank Drs. Kenji Omori and Jun Kotera of Tanabe-Seiyaku Pharmaceutical Co. Ltd. (Saitama, Japan) for kindly providing human PDE5A1 cDNA. We also thank Kennard Grimes for excellent technical assistance. Many reagents were obtained via the Vanderbilt University Diabetes Center Core facilities with the aid of Scott Wright, Kris Ellis, and Kennetra Price. DNA sequencing was performed by the Vanderbilt Ingram Cancer Center Sequencing Core Facility. Download .pdf (.09 MB) Help with pdf files
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