Identification of Residues Crucially Involved in the Binding of the Heme Moiety of Soluble Guanylate Cyclase
2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês
10.1074/jbc.m310141200
ISSN1083-351X
AutoresPéter Schmidt, Matthias Schramm, H. Schröder, Frank Wunder, Johannes-Peter Stasch,
Tópico(s)Hemoglobin structure and function
ResumoSoluble guanylate cyclase (sGC), a heterodimeric hemeprotein, is the only receptor for the biological messenger nitric oxide (NO) identified to date and is intimately involved in various signal transduction pathways. By using the recently discovered NO- and heme-independent sGC activator BAY 58-2667 and a novel cGMP reporter cell, we could distinguish between heme-containing and heme-free sGC in an intact cellular system. Using these novel tools, we identified the invariant amino acids tyrosine 135 and arginine 139 of the β1-subunit as crucially important for both the binding of the heme moiety and the activation of sGC by BAY 58-2667. The heme is displaced by BAY 58-2667 due to a competition between the carboxylic groups of this compound and the heme propionic acids for the identified residues tyrosine 135 and arginine 139. This displacement results in the release of the axial heme ligand histidine 105 and to the observed activation of sGC. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by this compound and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity. Soluble guanylate cyclase (sGC), a heterodimeric hemeprotein, is the only receptor for the biological messenger nitric oxide (NO) identified to date and is intimately involved in various signal transduction pathways. By using the recently discovered NO- and heme-independent sGC activator BAY 58-2667 and a novel cGMP reporter cell, we could distinguish between heme-containing and heme-free sGC in an intact cellular system. Using these novel tools, we identified the invariant amino acids tyrosine 135 and arginine 139 of the β1-subunit as crucially important for both the binding of the heme moiety and the activation of sGC by BAY 58-2667. The heme is displaced by BAY 58-2667 due to a competition between the carboxylic groups of this compound and the heme propionic acids for the identified residues tyrosine 135 and arginine 139. This displacement results in the release of the axial heme ligand histidine 105 and to the observed activation of sGC. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by this compound and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity. Soluble guanylate cyclase (sGC), 1The abbreviations used are: sGC, soluble guanylate cyclase; DEA/NO, diethylamine NONOate; NO, nitric oxide; ODQ, 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalin-1-one; PPIX, 3,18-divinyl-2,7,13,17-tetramethyl-porphine-8,12-dipropionic acid; YC-1, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole; BAY 58-2667, 4-[((4-carboxybutyl){2-[(4-phenethyl-benzyl)oxy]phenethyl}amino)methyl] benzoic acid; BAY 41-2272, 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine; CHO, Chinese hamster ovary cells; RLU, relative light unit(s); WT, wild type. an intracellular receptor for the ubiquitous biological messenger nitric oxide (NO), is a heterodimer consisting of an α- and β-subunit. The enzyme contains a prosthetic heme group that is bound via histidine 105 to the β-subunit of sGC. Activation of the enzyme upon binding of its physiological activator NO to the heme moiety catalyzes the conversion of GTP to the second messenger cGMP. cGMP regulates various effector systems such as phosphodiesterases, ion channels, and protein kinases, thereby modulating many physiological processes, including vasodilatation, neurotransmission, and platelet aggregation (1.Furchgott R. Angew. Chem. Int. Ed. Engl. 1999; 38: 1870-1880Crossref Google Scholar, 2.Ignarro L.J. Angew. Chem. Int. Ed. Engl. 1999; 38: 1882-1892Crossref Google Scholar, 3.Murad F. Angew. Chem. Int. Ed. Engl. 1999; 38: 1857-1868Crossref Google Scholar). The pathogenesis of various diseases, especially those of the cardiovascular system, has been linked to inappropriate activation of sGC (4.Moncada S. Palmer R.M. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar, 5.Hobbs A.J. Emerging Ther. Targets. 2000; 4: 735-749Crossref Scopus (16) Google Scholar, 6.Hobbs A.J. Br. J. Pharmacol. 2002; 136: 637-640Crossref PubMed Scopus (54) Google Scholar). The concept of NO-dependent sGC activation as a mechanism underlying antianginal action has been validated by the successful clinical use of NO-releasing drugs for more than a century (7.Megson I.L. Webb D.J. Expert Opin. Investig. Drugs. 2002; 11: 587-601Crossref PubMed Scopus (91) Google Scholar, 8.Napoli C. Ignarro L.J. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 97-123Crossref PubMed Scopus (203) Google Scholar). YC-1, an indazole derivative, was the first NO-independent activator of sGC discovered (9.Ko F.N. Wu C.C. Kuo S.C. Lee F.Y. Teng C.M. Blood. 1994; 84: 4226-4233Crossref PubMed Google Scholar, 10.Wu C.C. Ko F.N. Kuo S.C. Lee F.Y. Teng C.M. Br. J. Pharmacol. 1995; 116: 1973-1978Crossref PubMed Scopus (191) Google Scholar, 11.Friebe A. Schultz G. Koesling D. EMBO J. 1996; 15: 6863-6868Crossref PubMed Scopus (318) Google Scholar, 12.Mülsch A. Bauersachs J. Schäfer A. Stasch J.P. Kast R. Busse R. Br. J. Pharmacol. 1997; 120: 681-689Crossref PubMed Scopus (214) Google Scholar). Recently, the YC-1-related substance BAY 41-2272 has been identified as a novel, more specific, and more potent compound that stimulates sGC in a concentration-dependent manner and shows a strong synergism when combined with NO (13.Stasch J.P. Becker E.M. Alonso-Alija C. Apeler H. Dembowsky K Feurer A. Gerzer R. Minuth T. Perzborn E. Pleiss U. Schröder H. Schröder W. Stahl E. Steinke W. Straub A. Schramm M. Nature. 2001; 410: 212-215Crossref PubMed Scopus (490) Google Scholar). sGC activation through BAY 41-2272 requires the presence of the heme moiety, although a direct interaction with the heme-iron is not likely as suggested by spectroscopic studies (13.Stasch J.P. Becker E.M. Alonso-Alija C. Apeler H. Dembowsky K Feurer A. Gerzer R. Minuth T. Perzborn E. Pleiss U. Schröder H. Schröder W. Stahl E. Steinke W. Straub A. Schramm M. Nature. 2001; 410: 212-215Crossref PubMed Scopus (490) Google Scholar). In contrast to BAY 41-2272, another recently described non-NO-based sGC activator BAY 58-2667 stimulates not only the native sGC but, even more potently, the heme-deficient or oxidized form of the enzyme (14.Stasch J.P. Schmidt P. Alonso-Alija C. Apeler H. Dembowsky K. Haerter M. Heil M. Minuth T. Perzborn E. Pleiss U. Schramm M. Schröder H. Schröder W. Stahl E. Steinke W. Wunder F. Br. J. Pharmacol. 2002; 136: 773-783Crossref PubMed Scopus (269) Google Scholar, 15.Schmidt P. Schramm M. Schröder H. Stasch J.P. Eur. J. Pharmacol. 2003; 468: 167-174Crossref PubMed Scopus (91) Google Scholar) suggesting a novel mechanism of activation. The existence of these different types of non-NO-based sGC-activating compounds suggest that several unique allosteric regulatory sites are present on the enzyme and may open up new therapeutic avenues for cardiovascular diseases (6.Hobbs A.J. Br. J. Pharmacol. 2002; 136: 637-640Crossref PubMed Scopus (54) Google Scholar). The degree of sGC activation by these two different types of synthetic sGC activators as well as NO is dependent on the presence or oxidation state of the heme moiety. The involvement of this prosthetic group and the heme-binding domain in nearly all mechanisms of sGC activation (and inhibition) has been difficult to probe due to the lack of compounds capable of activating the heme-free enzyme. The only known function, ascribed to this domain for many years, was binding of the heme moiety to the β-subunit of sGC via the histidine 105 and its alteration upon NO binding (16.Wedel B. Humbert P. Harteneck C. Foerster J. Malkewitz J. Böhme E. Schultz G. Koesling D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2592-2596Crossref PubMed Scopus (233) Google Scholar, 17.Zhao Y. Schelvis J.P. Babcock G.T. Marletta M.A. Biochemistry. 1998; 37: 4502-4509Crossref PubMed Scopus (157) Google Scholar, 18.Martin E. Lee Y.C. Murad F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12938-12942Crossref PubMed Scopus (71) Google Scholar). As early as 1984, however, Ignarro and coworkers (19.Ignarro L.J. Ballot B. Wood K.S. J. Biol. Chem. 1984; 259: 6201-6207Abstract Full Text PDF PubMed Google Scholar) could show the importance of the two propionic acid groups of the heme moiety for its binding to the enzyme and postulated: "These propionic acid groups, which are ionized at pH 7.4, may form tight ion pairs with positively charged groups in guanylate cyclase and thereby contribute to the binding of porphyrins." Using the indazole BAY 41-2272, the aminodicarboxylic acid BAY 58-2667 and a novel cGMP reporter cell we were able to identify the two residues tyrosine 135 and arginine 139 of the β1-subunit of sGC as the postulated counterparts of the propionic acid groups of the heme moiety. Alteration of these amino acids resulted not only in the loss of the heme binding capacity of the enzyme but also in a reduction of the BAY 58-2667-induced sGC activation. Based on these findings we suggest a model of sGC activation with two high affinity binding sites for BAY 58-2667: one that is saturable at nanomolar concentrations and shows no direct interference with the heme moiety as described earlier (14.Stasch J.P. Schmidt P. Alonso-Alija C. Apeler H. Dembowsky K. Haerter M. Heil M. Minuth T. Perzborn E. Pleiss U. Schramm M. Schröder H. Schröder W. Stahl E. Steinke W. Wunder F. Br. J. Pharmacol. 2002; 136: 773-783Crossref PubMed Scopus (269) Google Scholar), and a second one that exhibits a direct competition between BAY 58-2667 and the prosthetic heme for the tyrosine 135 and the arginine 139. This competition results in the displacement of the heme moiety and as a consequence to the observed activation of sGC probably due to the release of the axial heme ligand histidine 105. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by BAY 58-2667 and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity. Compounds—BAY 58-2667 (4-[((4-carboxybutyl){2-[(4-phenethylbenzyl)oxy]phenethyl}amino)methyl] benzoic acid) and BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine) were synthesized as described previously (20.Alonso-Alija, C., Heil, M., Flubacher, D., Naab, P., Stasch, J. P., Wunder, F., Dembowsky, K, Perzborn, E., and Stahl, E. (2001) Patent WO-11978-A 2001.03.22Google Scholar, 21.Straub A. Stasch J.P. Alonso-Alija C. Benet-Buchholz J Ducke B. Feurer A. Fürstner C. Bioorg. Med. Lett. 2001; 11: 781-784Crossref PubMed Scopus (151) Google Scholar). DEA/NO (2-(N,N-diethylamino)diazenolate-2-oxide), ODQ (1H-(1,2,4)-oxadiazole[4,3-a]quinoxalin-1-one), and PPIX (3,18-divinyl-2,7,13,17-tetramethylporphine-8,12-dipropionicacid) were purchased from Alexis Biochemicals (San Diego, CA). All other chemicals of analytical grade were obtained from Sigma (Taufkirchen, Germany). Mutagenesis—The mutagenesis was performed using the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's protocols. The following primers were used to perform the desired mutations: βH105F: 5′-GCAGAACCTCGACGCCCTGTTCGACCACCTCGCCACC-3′; βY135F: 5′-GCAAAGGGCTCATTCTGCACTTCTACTCGGAAAGAGAGGGGC-3′; βY135A: 5′-GCAAAGGGCTCATTCTGCACGCCTACTCGGAAAGAGAGGGGC-3′; βR139L; 5′-CATTCTGCACTACTACTCGGAACTAGAGGGGCTTCAGGACATTG-3′; βR139A: 5′-CATTCTGCACTACTACTCGGAAGCAGAGGGGCTTCAGGACATTG-3′; βY135A+R139A: 5′-GCAAAGGGCTCATTCTGCACGCCTACTCGGAAGCAGAGGGGC-3′; and βY135F+R139L: 5′-GCAAAGGGCTCATTCTGCACTTCTACTCGGAACTAGAGGGGC-3′. The accuracy of the mutations was verified by sequencing (Invitek, Berlin, Germany). Generation of a cGMP Reporter Cell—To characterize sGC mutants in an intracellular environment, a cGMP reporter cell was constructed based on a method reported earlier (22.Wunder F. Lohrmann E. Hütter J. Stasch J.P. Hüser J. BMC Meeting Abstracts: 1st International Conference on cGMP. NO/sGC Interaction and Its Therapeutic Implications, Leipzig, Germany, June 14–16, 2003. 1. BioMed Central, London2003: 0059Google Scholar). Briefly, a CHO cell line expressing cytosolic aequorin was stably transfected with a plasmid coding for the cGMP-gated ion channel CNG2 under a zeocin resistance. Thereafter, zeocin-resistant clones were characterized for channel expression, and active clones were subcloned by the limited dilution technique. Selected clones were cultured in Dulbecco's modified Eagle's medium/F-12 with l-glutamine (Invitrogen, Carlsbad, CA), 1 mm sodium pyruvate, and 0.075% sodium bicarbonate, supplemented with 50 units/ml penicillin, 50 μg/ml streptomycin, 2.5 μg/ml amphotericin B, and 10% (v/v) inactivated fetal calf serum. Transient Transfection—cGMP readout cells were seeded on 96-well microtiter plates at a density of 20,000 cells per well and were cultured for 2 days at 37 °C and 5% CO2 to ensure confluent growth. Afterward, cells were cotransfected applying a transfection mixture containing 75 ng of α1- and 75 ng of β1-plasmid, 0.5 μl of Plus® reagent and 1 μl of LipofectAMINE® (Invitrogen) in 100 μl of Opti-MEM® serum-free medium (Invitrogen) according to the manufacturer's protocol. After 3 h at 37 °C the transfection medium was replaced with serum-containing medium, and cells were incubated for 48 h at 37 °C to ensure optimal expression of the WT-sGC and mutant-sGC. cGMP Readout—Cells were cultured and transiently transfected with WT-sGC or mutant-sGC as described above. 48 h after transfection medium was removed, and the transfected cells were incubated with calcium-free buffer (130 mm NaCl, 5 mm KCl, 20 mm HEPES, 1 mm MgCl2, 4.8 mm NaHCO3, pH 7.4) containing 0.83 μg/ml coelenterazine for 3 h at 37 °C. For the determination of the sGC activation profile, cells were incubated with various concentrations of sGC activators in a volume of 50 μl at 37 °C for 15 min. cGMP readout was initiated by application of buffer containing 10 mm CaCl2, and emitted light was measured as relative light units (RLU) in a light-tight box using a charge-coupled device camera. PPIX Reconstitution—To investigate whether the performed mutations influenced the heme binding capacity of the enzyme, WT-, βY135F-, and R139L-sGC were transiently expressed in CHO cells as described above. After 48 h, cells were harvested and centrifuged at 100,000 × g. The sGC-containing supernatant was applied to the sGC activity assay. Reconstitution was performed with increasing concentrations of PPIX in the presence of 0.5% Tween 20 and 1 μm BAY 41-2272 to amplify the PPIX-induced sGC activation (23.Friebe A. Koesling D. Mol. Pharmacol. 1998; 53: 123-127Crossref PubMed Scopus (210) Google Scholar, 24.Stasch J.P. Alonso-Alija C. Apeler H. Dembowsky K. Feurer A. Minuth T. Perzborn E. Schramm M. Straub A. Br. J. Pharmacol. 2002; 135: 333-343Crossref PubMed Scopus (134) Google Scholar). sGC Purification and Activity Assay—We expressed sGC by using a baculovirus/Sf9 expression system and measured enzyme activity as described earlier (25.Hoenicka M. Becker E.M. Apeler H. Sirichoke T. Schröder H. Gerzer R. Stasch J.P. J. Mol. Med. 1999; 77: 14-23Crossref PubMed Scopus (118) Google Scholar). Briefly, a bioreactor was filled with 2.5 liters of medium (SF900II, 10% fetal calf serum) and inoculated with SF9 cultures. The culture was incubated at 28 °C until a final density of 2 × 106 cells/ml. After infection with virus stocks containing both subunits of sGC, cells were incubated for further 88 h at 28 °C and pelleted by centrifugation. The pellet was harvested by sonication on ice, and sGC was purified as described (25.Hoenicka M. Becker E.M. Apeler H. Sirichoke T. Schröder H. Gerzer R. Stasch J.P. J. Mol. Med. 1999; 77: 14-23Crossref PubMed Scopus (118) Google Scholar). Spectroscopic Studies—UV-visible spectra were recorded from 300 to 600 nm on a Beckman DU 640 spectrophotometer as reported earlier (15.Schmidt P. Schramm M. Schröder H. Stasch J.P. Eur. J. Pharmacol. 2003; 468: 167-174Crossref PubMed Scopus (91) Google Scholar). 1 mm and 10 mm stock solutions of BAY 58-2667 were prepared in Me2SO and added to the samples to obtain a final concentration of 10 and 100 μm. ODQ was prepared as a 100 μm stock solution in MilliQ H2O and diluted to a final concentration of 10 μm. Samples containing 15 μg of sGC were incubated at 37 °C for 10 min in the absence or presence of BAY 58-2667 (10 and 100 μm) alone or combined with ODQ. Before the UV-visible spectra were recorded, the enzyme was separated by ion exchange chromatography from BAY 58-2667, ODQ, or free heme that might interfere with the measurement. Separation of sGC from Detergent—Separation of sGC from detergent and unbound heme was performed as previously described (26.Schmidt P. Schramm M. Schröder H. Stasch J.P. Protein Expr. Purif. 2003; 31: 42-46Crossref PubMed Scopus (17) Google Scholar). Briefly, sGC-containing samples were loaded onto ion exchange columns and washed once to remove any traces of detergent. Bound sGC was eluted with 60 μl of elution buffer (300 mm NaCl), and UV-visible spectra were recorded as described above. Sequence Alignments—The N-terminal 200 amino acids of the β1-subunit of sGC are sufficient to bind the prosthetic heme moiety with spectral characteristics comparable to those of the native sGC (27.Schelvis J.P. Zhao Y. Marletta M.A. Babcock G.T. Biochemistry. 1998; 37: 16289-16297Crossref PubMed Scopus (47) Google Scholar, 28.Marletta M.A. Karow D. Pellicena P. Kuriyan J. Pan D. Mathies R. BMC Meeting Abstracts: 1st International Conference on cGMP. NO/sGC Interaction and Its Therapeutic Implications, Leipzig, Germany, June 14–16, 2003. 1. BioMed Central, London2003Google Scholar). Based on this information, we initiated a BLAST search with this putative heme-binding domain. Various sGC α- and β-subunits from different species as well as predicted proteins of unknown function were found. A subsequent alignment of sequences known to bind heme identified invariant or conserved amino acids (Fig. 1). From these residues, the positively charged and polar amino acids βH105, βY135, and βR139 were chosen for site-directed mutagenesis as indicated by the asterisk. Screening sGC Mutants—For screening sGC mutants we constructed a novel cGMP reporter cell line based on a CHO cell stably transfected with a cGMP-dependent cation channel (CNG2) and cytosolic aequorin. After maximum activation of this reporter cell in a 96-well microtiter plate, a signal of about 2,500,000 RLUs in each well could be measured. To ensure that the measured sGC activity was within the linear range of the readout system the experimental conditions were chosen to obtain a maximum signal between 500,000 and 1,000,000 RLUs after maximal sGC activation (see Supplemental Material to Fig. 2). The different mutants of the sGC β1-subunit were constructed according to the results of the alignment (Fig. 1), verified by sequencing and expressed in the cGMP reporter cell line. WT-sGC—As shown in Fig. 2A CHO cells cotransfected with WT α1- and β1-cDNA exhibited the activation profile of heme-containing sGC. DEA/NO activated the transiently transfected reporter cells to a maximum of 26.7-fold with an EC50 of 27 nm (Fig. 3B; see Supplemental Material to Fig. 2). In addition, a concentration-dependent stimulation of 15.9-fold could be achieved by the NO-independent but heme-dependent sGC-stimulator BAY 41-2272 (Fig. 2A and Table I). In the presence of 10 nm DEA/NO, a concentration that exhibits only negligible effects on sGC activation (2.2-fold; Fig. 3A), the maximal activation of BAY 41-2272 was increased 60% (Fig. 2A and Table I). The NO- and heme-independent sGC activator BAY 58-2667 showed a concentration-dependent activation of the transiently transfected enzyme of 7.4-fold that was potentiated up to 25.6-fold in the presence of the sGC inhibitor ODQ (Fig. 2A and Table I).Fig. 3NO-induced activation of the cGMP-readout cells transiently transfected with WT- or mutated sGC in the presence of 10 nm (A) or 1 μm (B) DEA/NO. Data are mean ± S.E. from 5–15 independent experiments performed in quadruple. C shows the concentration-response curve of expressed WT-, βY135F-, and βR139L-sGC after incubation with increasing concentrations of PPIX in the presence of Tween 20 (0.5%) and BAY 41-2272 (1 μm). Data are mean ± S.E. from 3–5 independent experiments performed in duplicate. sGC activation is represented as x-fold compared with the transfected but not stimulated control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IStimulation of different sGC mutants Maximal stimulation factors (x-fold) and EC50 values in nm of WT-sGC and mutant-sGC after incubation with BAY 41-2272 (1 nm to 10 μm) and BAY 58-2667 (1 nm to 10 μm) in the absence and presence of DEA/NO (10 nm) or ODQ (10 μm). Data are mean ± S.E. of 5–16 independent experiments performed in quadruple.BAY 41-2272BAY 58-2667– NO+ 10 nm NO– ODQ+ 10 μm ODQEC50x-foldEC50x-foldEC50x-foldEC50x-foldWT607 ± 5915.9 ± 0.4386 ± 4425.4 ± 0.588.7 ± 127.43 ± 0.1416.8 ± 2.725.6 ± 0.5βH105FNDaND, not determined2.09 ± 0.08ND2.44 ± 0.1511.9 ± 3.47.32 ± 0.2011.0 ± 3.87.98 ± 0.27βY135FND1.63 ± 0.03ND3.04 ± 0.10471 ± 4533.9 ± 0.6366 ± 3233.8 ± 0.6βY135ANDNDNDND460 ± 936.60 ± 0.23264 ± 516.36 ± 0.17βR139LND1.75 ± 0.08ND2.91 ± 0.211725 ± 39821.9 ± 1.311011 ± 29421.1 ± 1.6βR139ANDNDNDND7000bExtrapolated from concentration response curve25bExtrapolated from concentration response curve7000bExtrapolated from concentration response curve25bExtrapolated from concentration response curveβYR→FLNDNDNDND8300bExtrapolated from concentration response curve25bExtrapolated from concentration response curve7000bExtrapolated from concentration response curve23bExtrapolated from concentration response curveβYR→AANDNDNDNDNDNDNDNDa ND, not determinedb Extrapolated from concentration response curve Open table in a new tab βH105F—The mutant βH105F was generated as a control that causes the expression of heme-free sGC. Indeed, the mutant βH105F exhibited the activation pattern expected of heme-free sGC: no measurable activation of the enzyme in the presence of DEA/NO (Fig. 3, A and B), a negligible activation (2.1-fold) after incubation with BAY 41-2272, not further elevated when combined with NO (Fig. 2B and Table I), and activation by heme-independent BAY 58-2667 (7.3-fold), which was not enhanced by the presence of ODQ (Fig. 2B and Table I). βY135F and βY135A—Neither mutants βY135F or βY135A were activated by DEA/NO (Fig. 3, A and B). Both mutations abolished nearly completely the enzyme activation by BAY 41-2272 alone or when combined with NO (Fig. 2, C and D, and Table I). In contrast, both mutants were responsive to BAY 58-2667 with similar EC50 values reaching a maximal activation of 34-fold and 6.6-fold, respectively (Table I). This activation was not further potentiated by additional ODQ (Fig. 2, C and D, and Table I). βR139L and βR139A—These mutations caused the loss of the responsiveness toward NO and BAY 41-2272 (Figs. 2E, 2F, 3A, 3B, and Table I). In contrast, both mutants could be activated by BAY 58-2667 with a minimal effective concentration of about 1 μm. βR139L achieved a maximal activation of 21.9-fold in the absence and 21.1-fold in the presence of ODQ. The exchange βR139A was not saturable within the tested concentration range of BAY 58-2667 (Fig. 2F). After incubation with 10 μm BAY 58-2667, an activation of 16.8-fold was determined increasing to 18.7-fold in the presence of ODQ (Fig. 2F). Based on the extrapolated concentration-response curves, EC50 values of about 7 μm were assumed. βY135F plus R139L and βY135A plus βR139A—The construct with both Y135F and R139L resulted in an enzyme that was insensitive to NO, BAY 41-2272, and the combination of both (Figs. 2G, 3A, 3B, and Table I). Incubation with 10 μm BAY 58-2667 led to an activation of 13.2-fold increasing to 16.0-fold upon addition of ODQ (Fig. 2G). A saturable activation could not be reached within the tested concentration range. According to the calculated concentration-response curves, EC50 values of about 8 μm and a maximal stimulation factor of 25 were assumed (Table I). The exchange of both residues with alanine abolished any sGC activation by NO, BAY 41-2272, and BAY 58-2667 within the tested concentration range (Figs. 2H, 3A, and 3B). PPIX Reconstitution—To explore the influence of the tyrosine 135 and the arginine 139 for the binding of the heme moiety, reconstitution studies with protoporphyrin IX (PPIX), which mimics the nitrosyl-heme complex, were performed with WT-, βY135F-, and βR139L-sGC (Fig. 3C). Due to its activating effect PPIX represents a useful tool to investigate sGC reconstitution (19.Ignarro L.J. Ballot B. Wood K.S. J. Biol. Chem. 1984; 259: 6201-6207Abstract Full Text PDF PubMed Google Scholar, 24.Stasch J.P. Alonso-Alija C. Apeler H. Dembowsky K. Feurer A. Minuth T. Perzborn E. Schramm M. Straub A. Br. J. Pharmacol. 2002; 135: 333-343Crossref PubMed Scopus (134) Google Scholar, 29.Ignarro L.J. Wood K.S. Wolin M.S. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2870-2873Crossref PubMed Scopus (200) Google Scholar). Reconstitution was performed in the presence Tween 20 for both the removal of the native heme moiety and to facilitate the subsequent reconstitution with PPIX (30.Foerster J. Harteneck C. Malkewitz J. Schultz G. Koesling D. Eur. J. Biochem. 1996; 240: 380-386Crossref PubMed Scopus (72) Google Scholar). Furthermore, BAY 41-2272 was added to the incubation buffer to amplify the sGC activation upon PPIX reconstitution (23.Friebe A. Koesling D. Mol. Pharmacol. 1998; 53: 123-127Crossref PubMed Scopus (210) Google Scholar, 24.Stasch J.P. Alonso-Alija C. Apeler H. Dembowsky K. Feurer A. Minuth T. Perzborn E. Schramm M. Straub A. Br. J. Pharmacol. 2002; 135: 333-343Crossref PubMed Scopus (134) Google Scholar). WT-sGC could be reconstituted with increasing concentrations of PPIX leading to a maximal sGC activation of 79.6-fold with an EC50 of 3.1 μm in the presence of 1 μm BAY 41-2272 (Fig. 3C). In contrast, 30 μm PPIX was required to achieve even a slight increase (3.1-fold) in the activity of the βR139L mutant. The βY135F exchange was not responsive even at the highest applied concentration of PPIX (Fig. 3C). Spectroscopic Studies—To explore potential interactions between the heme moiety of sGC and BAY 58-2667, the enzyme was incubated with BAY 58-2667 in the absence and presence of ODQ and separated subsequently by ion exchange chromatography. The native ferrous sGC showed the Soret band at 431 nm that was shifted to 392 nm after oxidizing the heme by addition of ODQ (Fig. 4A). Incubation of the native sGC with 10 μm BAY 58-2667 did not result in any shift of the Soret peak (Fig. 4B). Incubation of sGC with 10 μm BAY 58-2667 in the presence of ODQ led to the removal of the prosthetic heme moiety as did incubation with 100 μm BAY 58-2667 without additional ODQ (Fig. 4B). sGC Activity Assay—Purified WT-sGC was incubated with increasing concentrations of BAY 58-2667 from 100 pm to 200 μm in the absence and presence of 10 μm ODQ. As shown in Fig. 4C, a biphasic activation of sGC was observed in the absence of ODQ. The first step displayed a concentration-dependent activation of the enzyme from 1 nm to 50 nm with an EC50 of 3.6 nm followed by a phase of slight increase of sGC activation with increasing concentrations of BAY 58-2667 up to about 3 μm. Thereafter, a second phase of sGC activation started from 3 μm until the maximal solubility of this compound was reached (200 μm). In the presence of ODQ a sigmoidal concentration response curve with an EC50 of 9.6 nm was observed reaching a maximal specific activity of 19,050 nmol·min-1·mg-1. Here we report the identification of amino acids crucial for binding of the prosthetic heme moiety to sGC as well as for NO-independent sGC activation through BAY 58-2667. Based on these findings, we propose a model for the BAY 58-2667-induced activation of sGC summarized in Fig. 5. This model might also be useful to understand the NO-driven activation of the enzyme via a signal transmission triad consisting of the histidine 105, tyrosine 135, and arginine 139 located within the β1-subunit of sGC. These residues might be involved in the transduction of heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity. In the early 1980s, studies with different porphyrin derivatives suggested that the propionic acid groups of the porphyrin interact with basic residues of the enzyme (19.Ignarro L.J. Ballot B. Wood K.S. J. Biol. Chem. 1984; 259: 6201-6207Abstract Full Text PDF PubMed Google Scholar). More than two decades later our knowledge of the heme binding domain and the intramolecular signal transduction has advanced little due to the lack of any crystal structure of the enzyme (31.Mayer B. Koesling D. Trends Pharmacol. Sci. 2001; 22: 546-548Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Moreover, elucidating the function of this domain by mutagenesis studies failed due to the lack of compounds capable of activating the heme-free enzyme. Using a novel cGMP reporter cell line that obviated the need to purify sGC mutants together with the newly discovered heme-independent sGC activator BAY 58-2667 (14.Stasch J.P. Schmidt P. Alonso-Alija C. Apele
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