Specific Structural Requirements for the Inhibitory Effect of Thapsigargin on the Ca2+ ATPase SERCA

Abstract

Mutational analysis of amino acid residues lining the thapsigargin (TG) binding cavity at the interface of the membrane surface and cytosolic headpiece was performed in the Ca2+ ATPase (SERCA-1). Specific mutations such as F256V, I765A, and Y837A reduce not only the apparent affinity of the ATPase for TG but also the maximal inhibitory effect. The effect of mutations is dependent on the type and size of the substitute side chain, indicating that hydrophobic partitioning of TG and complementary molecular shapes are involved not only in binding but also in the inhibitory mechanism. A major factor determining the inhibitory effect of bound TG is its interference with conformational changes that are required for the progress of the ATPase cycle. Most prominent and specific is the TG interference with a wide displacement of the Phe-256 side chain that is associated with the E2 to E1·2Ca2+ transition. The specificity of the TG inhibitory mechanism is emphasized by the finding that the F256V mutation does not interfere at all with the effect of 2,5-di-(t-butyl)-hydroquinone, which is another SERCA inhibitor bound by hydrophobic partitioning. The specificity of the inhibitory mechanism is also emphasized by the observation that within the concentration range producing total inhibition of wild-type SERCA-1, TG produces a 4-fold stimulation of the P-glycoprotein (multidrug transporter) ATPase. Mutational analysis of amino acid residues lining the thapsigargin (TG) binding cavity at the interface of the membrane surface and cytosolic headpiece was performed in the Ca2+ ATPase (SERCA-1). Specific mutations such as F256V, I765A, and Y837A reduce not only the apparent affinity of the ATPase for TG but also the maximal inhibitory effect. The effect of mutations is dependent on the type and size of the substitute side chain, indicating that hydrophobic partitioning of TG and complementary molecular shapes are involved not only in binding but also in the inhibitory mechanism. A major factor determining the inhibitory effect of bound TG is its interference with conformational changes that are required for the progress of the ATPase cycle. Most prominent and specific is the TG interference with a wide displacement of the Phe-256 side chain that is associated with the E2 to E1·2Ca2+ transition. The specificity of the TG inhibitory mechanism is emphasized by the finding that the F256V mutation does not interfere at all with the effect of 2,5-di-(t-butyl)-hydroquinone, which is another SERCA inhibitor bound by hydrophobic partitioning. The specificity of the inhibitory mechanism is also emphasized by the observation that within the concentration range producing total inhibition of wild-type SERCA-1, TG produces a 4-fold stimulation of the P-glycoprotein (multidrug transporter) ATPase. Sesquiterpene lactone thapsigargin (TG), 1The abbreviations used are: TG, thapsigargin; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; DBHQ, 2,5-di-(t-butyl)hydroxybenzene; WT, wild-type; DBTG, debutanoyl TG; MOPS, 4-morpholinepropanesulfonic acid. derived from the plant Thapsia garganica (1Rasmussen U. Christensen B S. Sandberg F. Acta Pharm. Suec. 1978; 15: 133-140PubMed Google Scholar, 2Christensen S.B. Larsen I.K. Rasmuseen U. Christopherson C. J. Org. Chem. 1982; 47: 649-652Crossref Scopus (68) Google Scholar), is a potent and highly specific inhibitor of the endo- (3Thastrup O. Cullen P.J. Drobak P.K. Hanley M.R. Dawson A.P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (3010) Google Scholar) and sarcoplasmic reticulum (4Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar, 5Lytton J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar, 6Wictome M. Khan Y.M. East M.J. Lee A.G. Biochem. J. 1992; 283: 525-529Crossref PubMed Scopus (80) Google Scholar) Ca2+ ATPases (SERCA). Because of the role of intracellular Ca2+ stores in control and regulation of numerous cellular functions (7Berridge M.J. Bootman M.D. Lipp P. Nature. 1998; 395: 645-648Crossref PubMed Scopus (1780) Google Scholar, 8Carafoli E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1115-1122Crossref PubMed Scopus (678) Google Scholar), TG is used extensively as a pharmacological tool in physiological experimentation (9Treiman M. Caspersen C. Christansen S.B. Trends Pharmacol. Sci. 1998; 19: 131-135Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar, 10Raeymaekers L. Nilius B. Voets T. Van Baelen K. Vanoevelen J. Wuytack F. Novartis Found Symp. 2002; 246: 71-76Crossref PubMed Google Scholar). Furthermore, TG is being considered for targeted cancer treatment (11Denmeade S.R. Jakobsen C.M. Janssen S. Khan S.R. Garrett E.S. Lilja H. Christensen S.B. Isaacs J. J. Natl. Cancer Inst. 2003; 95: 990-1000Crossref PubMed Scopus (246) Google Scholar) because of its inhibitory effect on cell proliferation (12Ghosh T.K. Bian J.H. Short A.D. Rybak S.L. Gill D.L. J. Biol. Chem. 1991; 266: 24690-24697Abstract Full Text PDF PubMed Google Scholar). Inhibition of Ca2+ ATPase is observed at subnanomolar concentrations of TG and is attributed to drug binding to the enzyme with a 1:1 molar ratio (4Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar). TG induces stabilization of the ATPase protein in a state similar to that of the Ca2+-free enzyme (i.e. E2), facilitating formation of ordered arrays (13Sagara Y. Wade J.B. Inesi G. J. Biol. Chem. 1992; 267: 1286-1292Abstract Full Text PDF PubMed Google Scholar) for structural studies by electron microscopy and x-ray diffraction (14Zhang P. Toyoshima C. Yonekura K. Green N.M. Stokes D.L. Nature. 1998; 392: 835-839Crossref PubMed Scopus (265) Google Scholar, 15Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). Spectroscopic (16Hua S. Malak H. Lakowicz J.R. Inesi G. Biochemistry. 1995; 34: 5137-5142Crossref PubMed Scopus (16) Google Scholar), photolabeling (17Hua S. Inesi G. Biochemistry. 1997; 36: 11865-11872Crossref PubMed Scopus (22) Google Scholar), chimerization (18Norregaard A. Vilsen B. Andersen J.P. J. Biol. Chem. 1994; 269: 26598-26601Abstract Full Text PDF PubMed Google Scholar), and mutational studies (19Yu M. Zhang L. Rishi A. Khaedeer M. Inesi G. Hussain A. J. Biol. Chem. 1998; 273: 3542-3546Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 20Zhong L. Inesi G. J. Biol. Chem. 1998; 273: 12994-12998Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) suggested that the TG binding site resides near the M3 segment at the cytosolic membrane surface. TG was then clearly identified by diffraction studies (15Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar) in a cavity delimited by the M3, M5, and M7 transmembrane helices near the cytosolic surface of the membrane (Fig. 1). We report here a mutational study, including construction and characterization of 20 mutations within the region surrounding the TG binding pocket. The aim of this study was to evaluate specific structural features that may be involved in determining the ATPase affinity for TG and its inhibitory mechanism. The specificity of the TG inhibitory mechanism was further investigated with comparative experiments on the SERCA inhibitor 2,5-di-(t-butyl)-hydroxybenzene (DBHQ) (22Llopis J. Chow S.B. Kass G.E. Gahm A. Orrenius S Biochem. J. 1991; 277: 553-556Crossref PubMed Scopus (64) Google Scholar, 23Wictome M. Michelangeli F. Lee A.G. East J.M. FEBS Lett. 1992; 304: 109-113Crossref PubMed Scopus (89) Google Scholar) and on the ATPase of the P-glycoprotein drug transporter (24Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1929) Google Scholar, 25Al-Shawi M.K. Senior A.E. J. Biol. Chem. 1993; 268: 4197-4206Abstract Full Text PDF PubMed Google Scholar). Preparation of Membrane Vesicles—Native sarcoplasmic reticulum vesicles were obtained from rabbit skeletal muscle as described previously (26Eletr S. Inesi G. Biochim. Biophys. Acta. 1972; 282: 174-179Crossref PubMed Scopus (308) Google Scholar). PCR Mutagenesis and Protein Expression—The chicken fast muscle SERCA-1 cDNA (27Karin N.J. Kaprielian Z. Fambrough D.M. Mol. Cell. Biol. 1989; 9: 1978-1986Crossref PubMed Scopus (71) Google Scholar) was subcloned into the SV40-pAdlox vector for site-directed mutagenesis. Primers of 20-30 bp in length were synthesized for each individual mutation. These primers were utilized to hybridize DNA sequences internal to the flanking primers and were used for PCR mutagenesis by the overlap extension method as described by Ho et al. (28Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6851) Google Scholar). Briefly, two overlapping fragments containing the mismatched bases of the targeted sequence were amplified in separate PCRs. The reaction products were then mixed and amplified by PCR using both flanking primers. The mutant cassette was then exchanged with the corresponding cassette of wild-type cDNA in SV40-pAdlox. After DNA sequencing, the mutated cDNA was transfected into COS-1 cells for overexpression of protein under control of the SV40 promoter. COS-1 cell cultures and transfection methods were described by Sumbilla et al. (29Sumbilla C. Lu L. Lewis D.E. Inesi G. J. Biol. Chem. 1993; 268: 21185-21192Abstract Full Text PDF PubMed Google Scholar). Microsomal Preparation and Immunodetection of Expressed Protein—The microsomal fraction of transfected COS-1 cells was obtained by differential centrifugation of homogenized cells (29Sumbilla C. Lu L. Lewis D.E. Inesi G. J. Biol. Chem. 1993; 268: 21185-21192Abstract Full Text PDF PubMed Google Scholar). Immunodetection of expressed ATPase in the microsomal fraction was obtained by Western blotting using 9E10 monoclonal antibody against the c-myc tag of SERCA-1. WT human P-glycoprotein was expressed in yeast, purified, and reconstituted in liposomes as described by Figler et al. (30Figler R.A. Omote H. Nakamoto R.K. Al-Shawi M.K. Arch. Biochem. Biophys. 2000; 376: 34-46Crossref PubMed Scopus (87) Google Scholar). Synthesis of TG Analog Debutanoyl Thapsigargin—TG (2.0 mg = 3.1 μmol) was dissolved in methanol and 10 μl of triethylamine and then incubated with stirring at room temperature for about 5 h in a small capped tube to obtain selective cleavage of the ester bond at C-8. The reaction was followed by high pressure liquid chromatography analysis as described by Hua and Inesi (17Hua S. Inesi G. Biochemistry. 1997; 36: 11865-11872Crossref PubMed Scopus (22) Google Scholar). The yield of the product was ∼90%. The structure of TG and debutanoyl TG (DBTG) are shown in Fig. 2. Functional Studies—SERCA ATPase activity was assayed in a reaction mixture containing 20 mm MOPS, pH 7.0, 80 mm KCl, 3 mm MgCl2, 10 μm free Ca2+, 5 mm sodium azide, 50 nm Ca2+ ATPase, 3 μm ionophore A23187, 3 mm ATP, and 0-10,000 nm TG. Ca2+-independent ATPase activity was assayed in the presence of 2 mm EGTA without added Ca2+. The reaction was started at 37 °C by adding ATP, and samples were taken at serial times for determination of Pi (31Lanzetta P.A. Alvarez L.J. Reinsch P.S. Candia O.A. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1821) Google Scholar). The Ca2+-dependent activity was calculated by subtracting the Ca2+-independent ATPase from the total ATPase and was corrected to account for the level of expressed protein in each microsomal preparation as revealed by immunoactivity and with reference to microsomes obtained from COS-1 cells transfected with wild-type SERCA-1 cDNA. P-glycoprotein ATPase activity was measured in a reaction mixture that contained 40 mm Tris-Cl, pH 7.4, 10 mm Na-ATP, 15 mm MgCl2, 0.1 mm EGTA, 2 mm NaN3, 85 nm P-glycoprotein, 1% Me2SO, and TG as indicated. The incubation was carried out at 37 °C, and samples were taken at serial times for determination of Pi product by the method of Van Veldhoven and Mannaerts (32Van Veldhoven P.P. Mannaerts G.P. Anat. Biochem. 1987; 161: 45-48Crossref PubMed Scopus (632) Google Scholar) as modified by Al-Shawi et al. (33Al-Shawi M.K. Ketchum C.J. Nakamoto R.K. Biochemistry. 1997; 36: 12961-12969Crossref PubMed Scopus (64) Google Scholar). The experimental points were fitted as described by Al-Shawi et al. (34Al-Shawi M.K. Polar M.K. Omote H. Figler R.A. J. Biol. Chem. 2003; 278: 52629-52640Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Digestion of WT or mutant ATPase proteinase K was conducted at 25 °C in media containing 50 mm MOPS, pH 7.0, 50 mm NaCl, 0.6 mg of microsomal protein (containing recombinant ATPase)/ml and 0.02 mg of proteinase K/ml, in the presence or in the absence of Ca2+ (1 mm EGTA present). The reaction was quenched at serial times with trichloroacetic acid to yield a final 2.5% concentration. The quenched protein was then solubilized with 1% sodium dodecyl sulfate, 0.312 m Tris, pH 6.8, 3.75% sucrose, 1.25 mm β-mercaptoethanol, and 0.025% bromphenol blue and subjected to electrophoretic analysis (35Laemmli U.K. Beguin F. Gujer-Kellenberger G. J. Mol. Biol. 1970; 47: 69-85Crossref PubMed Scopus (290) Google Scholar). Transgenic Expression of WT and Mutant SERCA—Approximately 10% of the COS-1 cells transfected under our conditions exhibited exogenous SERCA gene expression and targeted the Ca2+ ATPase to the endoplasmic reticulum, as shown by immunofluorescent and microscopy Z. Sumbilla C. Lewis S. Inesi G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Google Scholar). the experiments expressed the WT enzyme and 20 from or mutations of 10 amino acid residues near the TG binding site at the interface of the region with the headpiece of the ATPase. the mutational amino acid residues to the TG The expression of SERCA are because of the of transfection and because of the presence of The were by Western and and with a preparation of the WT enzyme (Fig. The on were obtained from and the specific of the mutations on the of of mutations on the effect of ATPase TG concentration to of of not TG concentration to not in a Most of the were expressed with the WT ATPase, such as or F256V, or and Most the mutation and mutations expression is apparent that the presence of residues in with of the chain in the presence of Z. Sumbilla C. Lewis S. Inesi G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Google Scholar). all of the expressed high ATPase activity with for the and was the expression and the ATPase activity of the ATPase and of total ATPase activity of microsomal a Ca2+-independent that from the total to obtain the specific Ca2+-dependent ATPase. be out that the activity of all was corrected to the SERCA of the microsomes used in the with reference to the preparation of the WT ATPase. of Ca2+ ATPase activity were in or COS-1 is shown in Fig. that the WT Ca2+ ATPase is within the concentration range of TG, and total inhibition of the Ca2+-dependent activity is observed in the presence of μm TG. concentration range of TG for such as the ATPase inhibition TG concentrations are such as the maximal inhibition that be obtained by the TG concentration is (Fig. Furthermore, for a amino the inhibitory effect of TG on the type of F256V as with (Fig. The of mutations on the ATPase to TG are shown in the TG concentration producing a inhibition is as on were by the F256V and but were by the I765A, and WT and for a TG all of the experiments the inhibitory effect of TG is as a of TG. of the of the TG concentration. because of the high affinity and the concentrations of TG to the ATPase, the free TG concentration in our reaction may be the total concentration of the mutational on the affinity of the inhibitor for the a in the effect of on a mutant on which the TG concentration is only to the WT is a debutanoyl of TG affinity for SERCA (17Hua S. Inesi G. Biochemistry. 1997; 36: 11865-11872Crossref PubMed Scopus (22) Google Scholar). this the free drug concentration is to the total concentration because the ATPase in the reaction mixture is and the inhibitory effect of on WT ATPase is observed within the nm shown in Fig. the drug concentrations required to obtain inhibition of the mutant are two of is used of TG. that the experiments with TG may in the mutational on the affinity of the inhibitor for the ATPase. TG with a of the mechanism of considered that TG at the interface of the M3, M5, and M7 transmembrane may interfere with structural that are required for the progress of the ATPase cycle. of be derived from observation of the of ATPase by proteinase K S. T. K. M. H. Toyoshima C. FEBS Lett. PubMed Scopus Google Scholar). electrophoretic analysis of fragments by in the absence of Ca2+ a and a the presence of Ca2+, the but the is not in is attributed to a Ca2+-dependent conformational to which of the A with a on the within the the segment to the A (15Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). is shown in Fig. that TG such effect of Ca2+, as the is in the presence of Ca2+, and the ATPase Furthermore, a effect of TG is in the absence of Ca2+ indicating that TG of the E2 the enzyme to by Ca2+. to the of to that TG to the effect of Ca2+ on with proteinase K in such the obtained with the F256V and the effect of Ca2+ in the presence of TG (Fig. the the which does not interfere with the TG a to that obtained with WT enzyme (Fig. indicating that the TG inhibition mechanism is to interference with conformational changes that are required for the activity of the Ca2+ ATPase. is that the of obtained in the presence of Ca2+ and in the absence of TG 2 in each or the absence of both Ca2+ and TG in each are in WT and that the and E2 is not by the mutations that such a reduce the apparent affinity of the and for TG. of the to TG, such as a specificity for studies that inhibition by a of hydrophobic is to interference with conformational changes of the enzyme Biochim. Biophys. Acta. 2002; PubMed Scopus Google Scholar), TG and may to separate Y.M. M. East J.M. Lee A.G. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). We then the F256V mutation interfere with the inhibitory effect of We that effect on the WT enzyme and on the F256V (Fig. which is the mutant in the inhibitory effect of TG (Fig. and of TG specificity is its of inhibitory effect on ATPases J. Westlin M. Hanley M.R. J. Biol. Chem. 1991; 266: 17067-17071Abstract Full Text PDF PubMed Google Scholar). is not such specificity is because of of binding or of the bound We then the ATPase activity of the drug P-glycoprotein may be by TG. is shown in Fig. that a 4-fold of the P-glycoprotein ATPase is by TG with apparent of μm followed by of this stimulation at concentrations = as is observed for in this enzyme (34Al-Shawi M.K. Polar M.K. Omote H. Figler R.A. J. Biol. Chem. 2003; 278: 52629-52640Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). that TG with high affinity to ATPases of structural but its to or activity is dependent on the structure of the studies that TG resides in a cavity delimited by the M3, M5, and M7 near the cytosolic surface of the membrane and mutational analysis that Phe-256 and are for TG is apparent that the Phe-256 a complementary surface to TG, which is to the of The effect by the mutation is with by corresponding changes on the TG structure S.B. Larsen I.K. Rasmuseen U. Christopherson C. J. Org. Chem. 1982; 47: 649-652Crossref Scopus (68) Google Scholar). The may be because of a in the the Y837A is The role of is and the effect of its mutation is to structural The of mutations on binding affinity are not dependent as on of the side chain as on the type and size of the mutation of Phe-256 to is to and the mutation to is to because of the length and hydrophobic of the substitute These are with the that TG binding is by hydrophobic partitioning and complementary molecular shapes Biochim. Biophys. Acta. 2002; PubMed Scopus Google Scholar). TG binding to the SERCA enzyme only bond with (15Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). be out that the ATPase activity of the mutant is of the WT We then considered the apparent affinity of this mutant for TG may be a effect to a to formation of E2 and a state level of this the samples were with TG the ATPase and the of TG from the inhibitory site is as with 10 for the the of enzyme TG ATPase is the observed inhibition is in to the level of and the ATPase in of a effect of the mutation on the TG binding by mutations with the TG inhibitory in to of binding the of structural specificity not only for TG binding but also for the inhibitory mechanism. is prominent in the F256V mutant but also in the and Y837A mutations such as the not this effect structural near the TG binding is that TG with Phe-256 and the ATPase were to the (Fig. also with may be to by its side chain the experiments on proteinase K that mutational interference with the inhibitory effect of TG on activity (Fig. and and with conformational (Fig. in A of our experiments is the that the inhibitory effect of TG a specific mechanism that is not by DBHQ, which is another SERCA inhibitor bound in a hydrophobic Furthermore, our experiments that the specificity inhibitory effect of TG for as to is not to the of binding but to the effect of the bound the 4-fold stimulation of the P-glycoprotein ATPase activity observed in our experiments clearly that the to bound TG may be in the of inhibition or on the enzyme is in this that the binding site of the P-glycoprotein a conformational in ATPase to drug D.M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, D.M. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). is that the structural of high affinity binding of the in total inhibition or stimulation of in two our finding that TG the P-glycoprotein ATPase a of the by cell subjected to selective pressure with TG (19Yu M. Zhang L. Rishi A. Khaedeer M. Inesi G. Hussain A. J. Biol. Chem. 1998; 273: 3542-3546Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, S.R. Hussain A. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). was that the cells of SERCA expression with Phe-256 but also of the that a role in the of including overexpression of the SERCA as as overexpression of the transporter These may be in the of the of TG in targeted cancer treatment (11Denmeade S.R. Jakobsen C.M. Janssen S. Khan S.R. Garrett E.S. Lilja H. Christensen S.B. Isaacs J. J. Natl. Cancer Inst. 2003; 95: 990-1000Crossref PubMed Scopus (246) Google Scholar). We O. for the and Zhang for in the of this