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• W2007803373 abstract "The contraction of skeletal muscle is regulated by Ca2+ binding to troponin C, which results in an internal reorganization of the interactions within the troponin-tropomyosin complex. Troponin T is necessary for Ca2+-dependent inhibition and activation of actomyosin. Troponin T consists of an extended NH2-terminal domain that interacts with tropomyosin and a globular COOH-terminal domain that interacts with tropomyosin, troponin I, and troponin C. In this study we used recombinant troponin T and troponin I fragments to delimit further the structural and regulatory interactions with the thin filament. Our results show the following: (i) the NH2-terminal region of troponin T activates the actomyosin ATPase in the presence of tropomyosin; (ii) the interaction of the globular domain of troponin T with the thin filament blocks ATPase activation in the absence of Ca2+; and (iii) the COOH-terminal region of the globular domain anchors the troponin C-troponin I binary complex to troponin T through a direct Ca2+-independent interaction with the NH2-terminal region of troponin I. This interaction is required for Ca2+-dependent activation of the actomyosin ATPase activity. Based on these results we propose a refined model for the troponin complex and its interaction with the thin filament. The contraction of skeletal muscle is regulated by Ca2+ binding to troponin C, which results in an internal reorganization of the interactions within the troponin-tropomyosin complex. Troponin T is necessary for Ca2+-dependent inhibition and activation of actomyosin. Troponin T consists of an extended NH2-terminal domain that interacts with tropomyosin and a globular COOH-terminal domain that interacts with tropomyosin, troponin I, and troponin C. In this study we used recombinant troponin T and troponin I fragments to delimit further the structural and regulatory interactions with the thin filament. Our results show the following: (i) the NH2-terminal region of troponin T activates the actomyosin ATPase in the presence of tropomyosin; (ii) the interaction of the globular domain of troponin T with the thin filament blocks ATPase activation in the absence of Ca2+; and (iii) the COOH-terminal region of the globular domain anchors the troponin C-troponin I binary complex to troponin T through a direct Ca2+-independent interaction with the NH2-terminal region of troponin I. This interaction is required for Ca2+-dependent activation of the actomyosin ATPase activity. Based on these results we propose a refined model for the troponin complex and its interaction with the thin filament. Troponin (Tn) 1The abbreviations used are: Tn, troponin; TnC, troponin C; TnI, troponin I; TnT, troponin T; Tm, tropomyosin; wt, wild type; DTT, dithiothreitol; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis. and tropomyosin (Tm) mediate the Ca2+-dependent regulation of skeletal muscle contraction through control of conformational features of the actin-based thin filament (1Ohtsuki I. Maruyama K. Ebashi S. Adv. Protein Chem. 1986; 38: 1-67Crossref PubMed Scopus (204) Google Scholar, 2Zot A.S. Potter J.D. Annu. Rev. Biophys. Chem. 1987; 16: 535-559Crossref PubMed Scopus (446) Google Scholar, 3Holmes K.C. Biophys. J. 1995; 68: 2-7PubMed Google Scholar, 4Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (460) Google Scholar, 5Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (475) Google Scholar). Actin, Tm, and Tn are present in the thin filaments in a molar ratio of 7:1:1 (6Ebashi S. Ohtsuki I. Mihashi K. Cold Spring Harbor Symp. Quant. Biol. 1973; 37: 215-223Crossref Google Scholar, 7Potter J.D. Arch. Biochem. Biophys. 1974; 162: 436-441Crossref PubMed Scopus (165) Google Scholar, 8Yates L.D. Greaser M.L. J. Biol. Chem. 1983; 258: 5770-5774Abstract Full Text PDF PubMed Google Scholar). The binary troponin C-troponin I (TnC·TnI) complex confers little Ca2+ sensitivity to the actomyosin ATPase activity at physiological ratios of actin to Tn, is not stably assembled on the actin filament, and is not capable of activating the ATPase at high Ca2+ levels. Troponin T (TnT) is necessary for full Ca2+ sensitivity of the actomyosin ATPase (9Hitchcock S.E. Eur. J. Biochem. 1992; 52: 255-263Crossref Scopus (76) Google Scholar, 10Greaser M.L. Gergely J. J. Biol. Chem. 1971; 246: 4226-4233Abstract Full Text PDF PubMed Google Scholar, 11Malnic B. Reinach F.C. Eur. J. Biochem. 1994; 222: 49-54Crossref PubMed Scopus (14) Google Scholar, 12Potter J.D. Sheng Z. Pan B.-S. Zhao J. J. Biol. Chem. 1995; 270: 2557-2562Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13Farah C.S. Miyamoto C.A. Ramos C.H.I. Da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar) and for restoration of full velocity in sliding filament assays (14Fraser I.D.C. Marston S.B. J. Biol. Chem. 1995; 270: 7836-7841Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The mechanism through which TnT exerts its role in the regulatory function of the troponin complex is not fully understood. TnT interacts with TnC, TnI, and Tm and holds the TnC/TnI dimer in the thin filament irrespective of the Ca2+ concentration (9Hitchcock S.E. Eur. J. Biochem. 1992; 52: 255-263Crossref Scopus (76) Google Scholar, 15Potter J.D. Gergely J. Biochemistry. 1974; 13: 2697-2703Crossref PubMed Scopus (214) Google Scholar, 16Mak A.S. Smillie L.B. J. Mol. Biol. 1981; 149: 541-550Crossref PubMed Scopus (119) Google Scholar, 17Fisher D. Wang G. Tobacman L.S. J. Biol. Chem. 1995; 270: 25455-25460Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Each Tm dimer spans seven actins, strongly suggesting that the regulatory function of the troponin complex is transduced through Tm to the actin molecules. TnT is the troponin subunit that most strongly binds to Tm (1Ohtsuki I. Maruyama K. Ebashi S. Adv. Protein Chem. 1986; 38: 1-67Crossref PubMed Scopus (204) Google Scholar, 2Zot A.S. Potter J.D. Annu. Rev. Biophys. Chem. 1987; 16: 535-559Crossref PubMed Scopus (446) Google Scholar). Two separate sites of attachment for TnT on Tm have been identified. The first site is near the head-to-tail overlap of sequential Tm molecules along the filament (16Mak A.S. Smillie L.B. J. Mol. Biol. 1981; 149: 541-550Crossref PubMed Scopus (119) Google Scholar) and interacts with the T1 fragment of TnT (Fig. 1). This interaction is independent of Ca2+ binding to TnC (18Pearlstone J.R. Smillie L.B. J. Biol. Chem. 1982; 257: 10587-10592Abstract Full Text PDF PubMed Google Scholar). The second site is within fragment T2 of TnT (Fig. 1) which binds near residues 150–180 of the Tm molecule (16Mak A.S. Smillie L.B. J. Mol. Biol. 1981; 149: 541-550Crossref PubMed Scopus (119) Google Scholar, 19White S.P. Cohen C. Phillips Jr., G.N. Nature. 1987; 325: 826-828Crossref PubMed Scopus (180) Google Scholar). The T2fragment of TnT also interacts with TnC and TnI in vitro and to Tm in a Ca2+-sensitive manner in the presence of TnC (18Pearlstone J.R. Smillie L.B. J. Biol. Chem. 1982; 257: 10587-10592Abstract Full Text PDF PubMed Google Scholar). In this work we used different combinations of TnT and TnI fragments to map the regions involved in both structural and regulatory interactions present in the trimeric troponin complex within the thin filament in the presence and in the absence of calcium. Actin (20Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: 164-182Crossref PubMed Scopus (981) Google Scholar) and myosin (21Reinach F.C. Masaki T. Shafiq S. Obinata T. Fischman D.A. J. Cell Biol. 1982; 95: 78-84Crossref PubMed Scopus (90) Google Scholar) were prepared from the pectoralis major muscle of adult chickens. α-Tm (22Smillie L.B. Methods Enzymol. 1982; 85: 234-241Crossref PubMed Scopus (226) Google Scholar) was prepared from adult chicken heart muscle. Recombinant TnI and TnC were isolated as described (23Quaggio R.B. Ferro J.A. Monteiro P.B. Reinach F.C. Protein Sci. 1993; 2: 1053-1056Crossref PubMed Scopus (24) Google Scholar, 24Fujimori K. Sorenson M. Herzberg O. Moult J. Reinach F.C. Nature. 1990; 345: 182-184Crossref PubMed Scopus (87) Google Scholar). Deletion mutants of TnI were prepared as described (13Farah C.S. Miyamoto C.A. Ramos C.H.I. Da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). Chicken skeletal muscle TnT-3 cDNA (GenBankTM accession number M22156) (25Smillie L.B. Golosinska K. Reinach F.C. J. Biol. Chem. 1988; 263: 18816-18820Abstract Full Text PDF PubMed Google Scholar) was used as a template for site-directed mutagenesis (26Kunkel T.A. Roberts J.D. Zakour R.A. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4558) Google Scholar). An NdeI site was inserted at codon Met1 (13Farah C.S. Miyamoto C.A. Ramos C.H.I. Da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). M13mp18-TnT-3 was mutated with the oligonucleotide 5′-GTTATACCAGTAGCAGACTGA-3′ to change the codon Leu217 into a stop codon (underlined), with the oligonucleotide 5′-GGCGCAAGTAATTGAACATTGA-3′ to change the Pro192 codon into a stop codon, and with the oligonucleotide 5′-AGGCGCAAGCATATGAACATTGACC-3′ to introduce an NdeI site (underlined) at codons 192 and 193. The NdeI-EcoRI fragments of the M13mp18-TnT-3 containing a stop codon at positions 192 or 217 were cloned into the same restriction sites of the pET-3a (27Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar). These vectors express, respectively, the fragments TnT1–216 (the first 216 amino acids of TnT) and TnT1–191 (the first 191 amino acids of TnT). The small NdeI-EcoRI fragment (produced by the second NdeI site, inserted at positions 192 and 193) was subcloned in the same sites of pET-3a, producing a vector for the expression of the fragment TnT194–263 (amino acids 194–263). For the production of the fragment TnT157–263, the NcoI-BamHI fragment of the expression vector pET-TnT* (11Malnic B. Reinach F.C. Eur. J. Biochem. 1994; 222: 49-54Crossref PubMed Scopus (14) Google Scholar) was subcloned in the same sites of pET-3d (27Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar). Escherichia coli BL21(DE3) pLysS (27Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6005) Google Scholar) was used to express wild-type TnT, TnT1–216, TnT1–191, TnT157–263, and TnT194–263. Cultures (4 liters) of E. coliharboring the different plasmids grown in 2× YT were induced with isopropyl-1-thio-β-d-galactopyranoside (0.4 mm final) in mid-log phase (A 600 = 0.8) and incubated for 3 h at 37 °C. For the purification of wild-type TnT, TnT1–216, TnT1–191, and TnT157–263, the cells were recovered by centrifugation at 3,000 × g (15 min), resuspended in 100 ml of 50 mm Tris-Cl, pH 8.0, 1 mm EDTA, 6 murea, 1.4 mm β-mercaptoethanol and lysed in a French press at 16,000 p.s.i. The extract was centrifuged (109,200 ×g, 40 min), and the supernatant was loaded onto a DEAE-Sepharose Fast-Flow column (Pharmacia XK16/70), and retained proteins were eluted with a 0–600 mm NaCl gradient. The fractions containing TnT were pooled and dialyzed against 50 mm sodium acetate, pH 5.0, 1 mm EDTA, 6m urea, 1.4 mm β-mercaptoethanol and loaded onto a CM-Sepharose Fast-Flow XK16/40 column equilibrated with the same buffer. Proteins were eluted with a 0–600 mm NaCl gradient in the same buffer, dialyzed against 50 mm Tris, pH 8.0, 1 mm EDTA, 1 m KCl, 1 mm DTT and stored at −20 °C. For the purification of the TnT194–263 fragment the E. coli cells were resuspended in 50 mm Tris, pH 8.0, 1 mm EDTA, 1.4 mm β-mercaptoethanol, lysed in the French press at 16,000 p.s.i., and centrifuged at 75,800 × g (40 min). The supernatant was loaded into a CM-Sepharose Fast-Flow XK16/40 column equilibrated with the same buffer. The protein was eluted with a 0–500 mm NaCl gradient, dialyzed against 50 mm Tris, pH 8.0, 6 m urea, 1.4 mm β-mercaptoethanol and loaded into a DEAE-Sepharose Fast-Flow column. The protein eluted in the flow-through was dialyzed against 50 mm Tris, pH 8.0, 1 mm EDTA, 1 m KCl, 1 mm DTT and stored at −20 °C. One molar KCl is necessary to maintain the TnTs in solution at high concentrations (100 μm). TnT1–191 is soluble in lower salt concentrations. Protein concentrations were determined (28Hartree E.F. Anal. Biochem. 1972; 48: 422-427Crossref PubMed Scopus (4546) Google Scholar), and the samples were analyzed in 15% SDS-PAGE. The deletion fragments presented the expected molecular masses: 25.5 kDa (TnT1–216), 22.5 kDa (TnT1–191), 12.5 kDa (TnT157–263), and 8.5 kDa (TnT194–263). Tn subunits (20 μm final of each) were combined in a 1:1:1 molar ratio in 6 m urea, 1 m KCl, 50 mmCaCl2, 20 mm imidazole, pH 7.5, 1 mm DTT. Successive dialysis (4 °C, 12 h each) against the same buffer containing 4.6 m urea, 2m urea, no urea, 100 mm KCl, and no KCl were used to gradually reduce the urea and salt concentrations. After dialysis the reconstituted complexes were centrifuged (12,000 ×g, 10 min), and the supernatant was aliquoted and stored at −70 °C. The actomyosin ATPases were measured as described previously (11Malnic B. Reinach F.C. Eur. J. Biochem. 1994; 222: 49-54Crossref PubMed Scopus (14) Google Scholar, 13Farah C.S. Miyamoto C.A. Ramos C.H.I. Da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). Actin (4 μm), Tm (0.58 μm), Tn (concentrations are indicated in the figure legends), and myosin (0.2 μm) were combined on ice in 20 mm imidazole, pH 7.0, 60 mm KCl, 3.5 mm MgCl2, 1 mm DTT, 0.5 mm EGTA. A 6 mm CaCl2 solution was used to obtain the desired pCa (K Ca2+ EGTA = 1.9 × 10−7m). Reactions were initiated by adding 2 mm Na2ATP, pH 7.0, after equilibration at 25 °C. After 15 min inorganic phosphate was determined using a colorimetric assay (29Heinomen J.K. Lahti R.J. Anal. Biochem. 1981; 113: 313-317Crossref PubMed Scopus (788) Google Scholar). Tn binding to actin/Tm was analyzed using an ultracentrifugation assay. Before each assay, Tn subunits and complexes were centrifuged to remove insoluble material. Actin (20 μm), Tm (2.86 μm), and Tn (2.86 μm) were combined in 20 mm imidazole, pH 7.0, 60 mm NaCl, 3 mm MgCl2, 0.5 mm CaCl2, 2 mm β-mercaptoethanol (+Ca2+), or in the same buffer with 0.5 mm EGTA replacing the CaCl2 (−Ca2+). The mixtures were centrifuged at 315,000 × g for 10 min at 4 °C in a Beckman Optima TLX ultracentrifuge. The pellets were rinsed and resuspended in the original volume. Equivalent volumes of the mixture before centrifugation and of the supernatants and pellets after centrifugation were analyzed by 15% SDS-PAGE or 12.5% Tricine/SDS-PAGE (30Schaegger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10479) Google Scholar) for the small fragments TnT194–263and TnT157–263. Densitometric quantification was performed using a dual wavelength scanner (Shimadzu CS-9000) at 550 nm. Since TnT and TnI and their fragments have a low solubility at low ionic strength, control experiments in the absence of actin-Tm were performed to ensure that at these relatively low protein concentrations, all the Tn components were soluble. A schematic representation of the TnT mutants is shown in Fig. 1. The precise sites for the insertion of stop codons were chosen so that the predicted α-helices would not be interrupted (Fig. 1). The mutants were expressed as non-fusion proteins and purified to homogeneity. Whole Tn complexes containing wt-TnC, wt-TnI, and wt-TnT or the different TnT fragments were reconstituted from the isolated components. After reconstitution, the TnT mutants became soluble at low ionic strength at high protein concentrations, indicating that they were incorporated into stable complexes. When analyzed by gel filtration in the presence of Ca2+, all complexes eluted as a single peak (data not shown). In the absence of Ca2+, TnT1–216 was dissociated from the ternary complex, which suggests that residues 217–263 in the COOH-terminal region of TnT are necessary for the incorporation of TnT into the trimeric complex in the absence of Ca2+ (see below). We analyzed the regulatory properties of complexes containing the different TnT mutants (Fig. 2 A). Under physiological molar ratios of actin, Tm, and Tn (7:1:1), we confirmed that the control Tn complex containing wt-TnT confers full Ca2+ sensitivity to actomyosin ATPase, i.e. it inhibits the ATPase activity in the absence of Ca2+ and activates the ATPase activity in the presence of Ca2+ (10Greaser M.L. Gergely J. J. Biol. Chem. 1971; 246: 4226-4233Abstract Full Text PDF PubMed Google Scholar) (activation is defined as the ability of troponin, in the presence of Ca2+, to increase the actomyosin/tropomyosin ATPase activity to levels above its activity in the absence of troponin). We also confirmed that the binary TnI·TnC complex was not able to inhibit or to activate the ATPase activity at physiological ratios of actin to troponin (Fig. 2 A) (10Greaser M.L. Gergely J. J. Biol. Chem. 1971; 246: 4226-4233Abstract Full Text PDF PubMed Google Scholar, 31Hitchcock S.E. Huxley H.E. Szent-Gyorgyi A. J. Mol. Biol. 1973; 80: 825-836Crossref PubMed Scopus (101) Google Scholar). Deletions of the COOH-terminal region of TnT or deletion of the T1region reduces the inhibitory function of the troponin complex (Fig. 2 A): TnT1–216 and TnT157–263complexes inhibit the ATPase activity to about 60% and TnT1–191 complex to about 75%, whereas the wt·TnT complex inhibits to about 35%. The complex containing TnT194–263 conferred no Ca2+ sensitivity to the actomyosin ATPase, a behavior identical to the TnI·TnC binary complex (Fig. 2 A). Since the partial inhibition observed for the TnT mutant complexes could be explained by a lower affinity for the thin filament, we performed ATPase experiments using increasing molar ratios of the troponin complex to actin. We observed full inhibitory activity of complexes containing TnT1–191, TnT1–216, and TnT157–263 at actin:troponin molar ratios of 4:7, 3:7, and 3:7 respectively (data not shown). These results imply that the region shared by these mutants, namely amino acids 157–216, bind to the thin filament and may have an inhibitory role. The complexes containing TnT1–216, TnT157–263, and TnT194–263 were not capable of activating the ATPase activity (Fig. 2 A) even when increasing ratios of Tn to actin were used (data not shown). The complex containing TnT1–191 was the only one capable of activating the ATPase activity like wt-Tn in the presence of Tm (Fig. 2 A). This suggests that the NH2-terminal end of TnT (residues 1–191) contains the region responsible for the activation of the ATPase activity. The evidence presented so far suggests that regions 1–191, 157–216, and 217–263 of TnT participate, respectively, in activation, inhibition/thin filament binding, and TnI/TnC binding. The experiments described below were designed to test and essentially confirm these hypotheses. The direct effects of the different regions of TnT on the inhibition and activation of the ATPase activity was analyzed in the presence of Tm but in the absence of TnI and TnC (Fig. 2 B). TnT1–191 is able to activate the ATPase activity, whereas wt-TnT and the mutants TnT157–263 and TnT194–263 are not. TnT1–216, which is not able to activate the ATPase activity in the context of the ternary complex (Fig. 2 A), is able to activate it in the absence of TnC and TnI, although to a lesser extent than TnT1–191. These results indicate that residues 191–263 of TnT are blocking the ability of region 1–191 to activate the ATPase activity and may explain why no activation is observed with isolated full-length TnT. Since full-length TnT alone does not activate the ATPase, but does so in the presence of TnI/TnC, we determined which regions within TnC/TnI are required for activation. We used TnI103–182, which binds to TnC-Ca2+ and is capable of inhibiting the ATPase activity but does not bind to TnT, and the TnI1–98 mutant, which binds to TnT and TnC but shows no inhibitory ability (13Farah C.S. Miyamoto C.A. Ramos C.H.I. Da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). The regulatory properties of the ternary complexes containing TnT, TnC, and the different TnI fragments are shown in Fig. 3 A. Although the complex containing TnI103–182 inhibited the ATPase activity as well as the complex containing TnI, it was not able to activate the ATPase activity. The complex containing TnI1–98 activates the ATPase to the same extent as the complex containing wt-TnI (Fig. 3 A). This activation was independent of the Ca2+ concentration, although slightly higher levels of activation were produced in the absence of Ca2+ as observed previously (13Farah C.S. Miyamoto C.A. Ramos C.H.I. Da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). These results indicate that the NH2-terminal region of TnI but not the COOH-terminal region is necessary for the activation of the ATPase activity. Next we analyzed if the NH2-terminal half of TnI is sufficient to activate the ATPase activity or if TnC is also required. The ATPase activity in the presence of filaments containing the different TnT mutants was titrated with increasing amounts of TnI1–98 in the absence of TnC (Fig. 3 B). TnI1–98 in combination with wt-TnT does not activate the ATPase in the absence of TnC. TnI1–98 in combination with TnT157–263 was also not able to activate. The effects of TnT1–191 and TnT1–216 on the ATPase activity were not altered by the addition of TnI1–98. These results indicate that TnI1–98 requires TnC to produce activation with wt-TnT. In conclusion, our ATPase results show that residues 1–191 of TnT contain an activation domain, whereas the region between residues 191 and 263 has an inhibitory effect. Thus activation can only be observed with intact TnT in the presence of TnC/TnI and Ca2+. Furthermore, the NH2-terminal regions of TnI in conjunction with TnC are necessary for activation by the ternary complex. The interactions between TnT fragments, TnI and TnC, were analyzed in an actin/tropomyosin co-sedimentation assay. The TnC·TnI complex is partially retained in the thin filament in the absence of Ca2+ but remains soluble if Ca2+ is present (Fig. 4 A). In the presence of wt-TnT, all three components of the Tn complex remained in the thin filament, irrespective of the Ca2+ concentration (Fig. 4 B) (15Potter J.D. Gergely J. Biochemistry. 1974; 13: 2697-2703Crossref PubMed Scopus (214) Google Scholar, 31Hitchcock S.E. Huxley H.E. Szent-Gyorgyi A. J. Mol. Biol. 1973; 80: 825-836Crossref PubMed Scopus (101) Google Scholar). If trimeric complexes containing TnT1–216 and TnT1–191 are incubated with actin/Tm, these two TnT mutants remained associated with the thin filament in the presence or absence of Ca2+ but were not able to retain the TnC/TnI dimer in the thin filament in the presence of Ca2+ (Fig. 4, C and D). These controls confirm the previous observations that TnT is required to anchor the TnC·TnI complex to the thin filament in the presence of Ca2+ (15Potter J.D. Gergely J. Biochemistry. 1974; 13: 2697-2703Crossref PubMed Scopus (214) Google Scholar, 31Hitchcock S.E. Huxley H.E. Szent-Gyorgyi A. J. Mol. Biol. 1973; 80: 825-836Crossref PubMed Scopus (101) Google Scholar) and that only the T2 region of TnT interacts with the TnC/TnI dimer (16Mak A.S. Smillie L.B. J. Mol. Biol. 1981; 149: 541-550Crossref PubMed Scopus (119) Google Scholar, 32Heeley D.H. Golosinska K. Smillie L.B. J. Biol. Chem. 1987; 262: 9971-9978Abstract Full Text PDF PubMed Google Scholar). These results specifically implicate the COOH-terminal half of T2(residues 217–263) in this interaction. It has been previously demonstrated that the NH2-terminal region of TnI (TnI1–98) is required for the incorporation of TnT into the ternary complex (12Potter J.D. Sheng Z. Pan B.-S. Zhao J. J. Biol. Chem. 1995; 270: 2557-2562Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 13Farah C.S. Miyamoto C.A. Ramos C.H.I. Da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). Fig. 4 E shows that the binary TnC·TnI1–98 complex does not associate with the thin filament in the absence of TnT irrespective of the Ca2+ concentration. In contrast, complexes containing TnC·TnI1–98·wt-TnT are always associated with the thin filament (Fig. 4 F). Since TnT1–216 and TnT1–191 lack residues 216–263 which interact with the TnC·TnI binary complex, they were not expected to, and indeed did not, retain TnI1–98/TnC bound to the thin filament (Fig. 4, G and H). Control complexes containing TnI103–182 (which does not bind TnT) were analyzed. TnI103–182 remained associated with the filament in the absence of Ca2+ and was removed by TnC in the presence of Ca2+, both in the presence and absence of TnT (13, data not shown). These findings demonstrate that the NH2-terminal region of TnI interacts with the region between residues 216 and 263 of TnT and that this interaction is the major point of calcium-independent anchoring of the TnC/TnI dimer to TnT. Since isolated TnC has been shown to interact with TnT in the presence of Ca2+ (reviewed in Refs. 1Ohtsuki I. Maruyama K. Ebashi S. Adv. Protein Chem. 1986; 38: 1-67Crossref PubMed Scopus (204) Google Scholar, 2Zot A.S. Potter J.D. Annu. Rev. Biophys. Chem. 1987; 16: 535-559Crossref PubMed Scopus (446) Google Scholar, and 5Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (475) Google Scholar), we analyzed the binding of the binary TnC·TnT complex (without TnI) to actin/Tm. The amount of TnC associated with the thin filaments was measured as a function of total TnC added in the presence or absence of TnI (Fig. 5). In the absence of TnI, a 10-fold excess TnC (30 μm) had to be used in the sedimentation assay to obtain approximately a 1:1 ratio of TnC:TnT bound to the filament (Fig. 5). In the presence of TnI, a 2-fold excess of TnC (6 μm) was sufficient. This suggests that there are two different sites of interaction between TnC and the thin filament in the presence of Ca2+. The first, through binding to TnI which in turn binds TnT, is a strong binding site (reviewed in Refs. 1Ohtsuki I. Maruyama K. Ebashi S. Adv. Protein Chem. 1986; 38: 1-67Crossref PubMed Scopus (204) Google Scholar, 2Zot A.S. Potter J.D. Annu. Rev. Biophys. Chem. 1987; 16: 535-559Crossref PubMed Scopus (446) Google Scholar, and 5Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (475) Google Scholar). The second, through direct interaction of TnC with TnT, is much weaker. In the presence and absence of Ca2+ the affinity of the TnT194–263·TnC·TnI complex for the thin filament is reduced (Fig. 6 B) when compared with the affinity of the TnT157–263·TnI·TnC complex (Fig. 6 A). This result suggests that a binding site to actin/Tm may reside in the NH2-terminal region of TnT157–263 (residues 157–191) and that this binding is influenced by Ca2+ binding to TnC. When the control TnT157–263·TnC complex was incubated with actin/Tm in the absence of Ca2+, TnT157–263 was incorporated into the filament while TnC remained in solution (Fig. 6 C). This confirms previous observations that TnT157–263 itself presents a binding site to actin/Tm (16Mak A.S. Smillie L.B. J. Mol. Biol. 1981; 149: 541-550Crossref PubMed Scopus (119) Google Scholar,32Heeley D.H. Golosinska K. Smillie L.B. J. Biol. Chem. 1987; 262: 9971-9978Abstract Full Text PDF PubMed Google Scholar). In the presence of Ca2+, both subunits remained in the supernatant indicating that TnC is able to remove TnT157–263 (TnT2) from the thin filament in the presence of Ca2+ (Fig. 6 C). Even though TnT2 fragment alone is able to interact with Tm (18Pearlstone J.R. Smillie L.B. J. Biol. Chem. 1982; 257: 10587-10592Abstract Full Text PDF PubMed Google Scholar) and with actin/Tm, it is still not clear how strong this interaction is in the context of the ternary troponin complex. Our results do not rule out the possibility that the interaction of TnT157–263 with the thin filament in the absence of Ca2+ is mainly mediated by TnI, which itself binds to the thin filament. To address this question we investigated how the interaction of TnT157–263 with the thin filament is modulated by the TnI fragments (Fig. 6, D and E). The TnT157–263·TnI1–98·TnC and TnT157–263·TnI103–182·TnC complexes were reconstituted, and their ability to be incorporated into the thin filament was analyzed. TnI103–182 binds actin/Tm and TnI1–98 does not (13Farah C.S. Miyamoto C.A. Ramos C.H.I. Da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). The TnT157–263·TnI1–98·TnC complex was not incorporated into the filament irrespective of the Ca2+ concentration (Fig. 6 D). When the trimeric TnT157–263·TnI103–182·TnC complex was incubated with actin/Tm in the absence of Ca2+, TnI103–182 and TnT157–263 were incorporated into the filament, whereas TnC remained in solution (Fig. 6 E). In the presence of Ca2+ the three subunits remained in solution (Fig. 6 E). These results indicate that although Ca2+ binding to TnC was able to remove both TnT157–263 and TnI103–182 from the filament, the interaction of TnI1–98/TnC with TnT reduces the affinity of TnT157–263 for the thin filament irresp" @default.
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• W2007803373 title "Regulatory Properties of the NH2- and COOH-terminal Domains of Troponin T" @default.
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• W2007803373 doi "https://doi.org/10.1074/jbc.273.17.10594" @default.
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