Mapping the Interacting Regions between Troponins T and C

Muscular contraction is triggered by an increase in calcium concentration, which is transmitted to the contractile proteins by the troponin complex. The interactions among the components of the troponin complex (troponins T, C, and I) are essential to understanding the regulation of muscle contraction. While the structure of TnC is well known, and a model for the binary TnC·TnI complex has been recently published (Tung, C.-S., Wall, M. E., Gallagher, S. C., and Trewhella, J. (2000)Protein Sci. 9, 1312–1326), very little is known about TnT. Using non-denaturing gels and NMR spectroscopy, we have analyzed the interactions between TnC and five peptides from TnT as well as how three TnI peptides affect these interactions. Rabbit fast skeletal muscle peptide TnT-(160–193) binds to TnC with a dissociation constant of 30 ± 6 μm. This binding still occurs in the presence of TnI-(1–40) but is prevented by the presence of TnI-(56–115) or TnI-(96–139), both containing the primary inhibitory region of TnI. TnT-(228–260) also binds TnC. The binding site for TnT-(160–193) is located on the C-terminal domain of TnC and was mapped to the surface of TnC using NMR chemical shift mapping techniques. In the context of the model for the TnC·TnI complex, we discuss the interactions between TnT and the other troponin subunits. Muscular contraction is triggered by an increase in calcium concentration, which is transmitted to the contractile proteins by the troponin complex. The interactions among the components of the troponin complex (troponins T, C, and I) are essential to understanding the regulation of muscle contraction. While the structure of TnC is well known, and a model for the binary TnC·TnI complex has been recently published (Tung, C.-S., Wall, M. E., Gallagher, S. C., and Trewhella, J. (2000)Protein Sci. 9, 1312–1326), very little is known about TnT. Using non-denaturing gels and NMR spectroscopy, we have analyzed the interactions between TnC and five peptides from TnT as well as how three TnI peptides affect these interactions. Rabbit fast skeletal muscle peptide TnT-(160–193) binds to TnC with a dissociation constant of 30 ± 6 μm. This binding still occurs in the presence of TnI-(1–40) but is prevented by the presence of TnI-(56–115) or TnI-(96–139), both containing the primary inhibitory region of TnI. TnT-(228–260) also binds TnC. The binding site for TnT-(160–193) is located on the C-terminal domain of TnC and was mapped to the surface of TnC using NMR chemical shift mapping techniques. In the context of the model for the TnC·TnI complex, we discuss the interactions between TnT and the other troponin subunits. troponin C troponin I troponin T C domain (residues 88–162) of recombinant chicken skeletal troponin C N domain (residues 1–90) of recombinant chicken skeletal troponin C heteronuclear single-quantum coherence Skeletal muscle contraction is regulated by troponin and tropomyosin located in the thin filament. Contraction begins with the binding of Ca2+ to the troponin complex, triggering conformational changes that are propagated to tropomyosin and actin. The troponin complex is constituted by a Ca2+-binding subunit, troponin C (TnC),1an inhibitory subunit, troponin I (TnI), and a tropomyosin-binding subunit, troponin T (TnT). When the muscle is relaxed, TnI inhibits myosin ATPase activity. The smallest region of TnI capable of this inhibition is known as the inhibitory region, or the inhibitory peptide (residues 96–115). Upon binding of Ca2+, TnC undergoes conformational changes and removes the inhibition by TnI. TnT binds to TnI, TnC, and tropomyosin, anchoring the troponin complex to the thin filament and propagating the conformational changes (for reviews, see Refs. 1Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-559Crossref PubMed Scopus (446) Google Scholar, 2Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (475) Google Scholar, 3Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (460) Google Scholar). The interactions among the three components in the troponin complex as well as how they are affected by the presence of calcium are essential for understanding the regulation of muscle contraction. The structure of skeletal TnC is well known and has been determined by both x-ray crystallography (4Herzberg O. James M.N.G. J. Mol. Biol. 1988; 203: 761-779Crossref PubMed Scopus (291) Google Scholar, 5Satyshur K.A. Rao S.T. Pyzalska D. Drendel W. Greaser M. Sundaralingam M. J. Biol. Chem. 1988; 263: 1628-1647Abstract Full Text PDF PubMed Google Scholar, 6Houdusse A. Love M.L. Dominguez R. Grabarek Z. Cohen C. Structure. 1997; 5: 1695-1711Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) and nuclear magnetic resonance (NMR) spectroscopy (7Slupsky C.M. Sykes B.D. Biochemistry. 1995; 34: 15953-15964Crossref PubMed Scopus (185) Google Scholar). NMR and crystallographic data provided some structural information on the interaction between TnC and fragments of TnI (8Campbell A.P. Sykes B.D. J. Mol. Biol. 1991; 222: 405-421Crossref PubMed Scopus (73) Google Scholar, 9McKay R.T. Tripet B.P. Hodges R.S. Sykes B.D. J. Biol. Chem. 1997; 272: 28494-28500Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 10McKay R.T. Pearlstone J.R. Corson D.C. Gagné S.M. Smillie L.B. Sykes B.D. Biochemistry. 1998; 37: 12419-12430Crossref PubMed Scopus (49) Google Scholar, 11Vassylyev D.G. Takeda S. Wakatsuki S. Maeda K. Maeda Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4847-4852Crossref PubMed Scopus (193) Google Scholar, 12Hernández G. Blumenthal D.K. Kennedy M.A. Unkefer C.J. Trewhella J. Biochemistry. 1999; 38: 6911-6917Crossref PubMed Scopus (20) Google Scholar, 13Li M.X. Spyracopoulos L. Sykes B.D. Biochemistry. 1999; 38: 8289-8298Crossref PubMed Scopus (246) Google Scholar, 14Mercier P. Li M.X. Sykes B.D. Biochemistry. 2000; 39: 2902-2911Crossref PubMed Scopus (49) Google Scholar). A model for the structure of the skeletal binary TnC·TnI complex was recently built based on a number of binding and activity assays such as cross-linking, fluorescent resonance energy transfer (FRET), and neutron scattering data as well as the available structural information (15Tung C.-S. Wall M.E. Gallagher S.C. Trewhella J. Protein Sci. 2000; 9: 1312-1326Crossref PubMed Scopus (43) Google Scholar). Despite the amount of information on the interactions between TnC and TnI that has emerged in the last few years, not much is known about the structure of TnT, either isolated or interacting with another troponin subunit. Rabbit skeletal TnT yields two fragments when digested by chymotrypsin. The N-terminal fragment, known as T1, binds to tropomyosin, while the C-terminal fragment, T2, binds TnI and TnC (for reviews, see Refs. 3Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (460) Google Scholar and 16Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (256) Google Scholar). Although TnI by itself inhibits myosin ATPase and TnC can remove the inhibition, TnT is necessary for the Ca2+-dependent regulation of myosin ATPase by the troponin complex. Direct interaction of TnT with TnC is responsible for this calcium-sensitizing effect (17Potter 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). Herein we show that two TnT peptides, containing residues 160–193 and 228–260, bind to TnC and that other peptides in the T2 region do not bind TnC. We also show that two TnI peptides containing the inhibitory region compete with TnT-(160–193) for TnC, while the first 40 residues of TnI bind to TnC simultaneously with TnT-(160–193). Using two-dimensional 1H,15N NMR spectroscopy to follow the titration of TnC with the peptide TnT-(160–193), we calculated the binding constant for this peptide and mapped the binding region on the surface of TnC. We show this mapping on the model of the binary TnC·TnI complex and discuss the competition results in this context. The expression and purification of recombinant chicken skeletal TnC, NTnC, CTnC, 15NTnC, and 13C,15NCTnC were performed as described previously (14Mercier P. Li M.X. Sykes B.D. Biochemistry. 2000; 39: 2902-2911Crossref PubMed Scopus (49) Google Scholar, 18Slupsky C.M. Kay C.M. Reinach F.C. Smillie L.B. Sykes B.D. Biochemistry. 1995; 34: 7365-7375Crossref PubMed Scopus (61) Google Scholar, 19Gagné S.M. Tsuda S. Li M.X. Chandra M. Smillie L.B. Sykes B.D. Protein Sci. 1994; 3: 1961-1974Crossref PubMed Scopus (176) Google Scholar). The rabbit fast skeletal muscle TnI and TnT peptide analogs were synthesized by solid phase peptide synthesis methodology and purified by reverse phase high performance liquid chromatography, as described (20Wagschal K. Tripet B. Hodges R.S. J. Mol. Biol. 1999; 285: 785-803Crossref PubMed Scopus (78) Google Scholar, 21Tripet B.P. Van Eyk J.E. Hodges R.S. J. Mol. Biol. 1997; 271: 728-750Crossref PubMed Scopus (184) Google Scholar, 22Ngai S.-M. Hodges R.S. J. Biol. Chem. 1992; 267: 15715-15720Abstract Full Text PDF PubMed Google Scholar, 23Sereda T.J. Mant C.T. Quinn A.M. Hodges R.S. J. Chromatogr. 1993; 646: 17-30Crossref PubMed Scopus (89) Google Scholar). TnT residue numbers correspond to isoform TnT2f (24Briggs M.M. Schachat F. J. Mol. Biol. 1989; 206: 245-249Crossref PubMed Scopus (42) Google Scholar). The peptide sequences were: TnT-(151–170), acetyl-SMGANYSSYLAKADQKRGKK-NH2; TnT-(160–193), acetyl-LAKADQKRGKKQTAREMKKKILAERRKPLNIDHL-NH2; TnT-(181–215), acetyl-LAERRKPLNIDHLSDEKLRDKAKELWDTLYQLETD-NH2; TnT-(209–243), acetyl-LYQLETDKFEFGEKLKRQKYDIMNVRARVEMLAKF-NH2; TnT-(228–260), acetyl-YDIMNVRARVEMLAKFSKKAGTTAKGKVGGRWK-NH2; TnI-(1–40), acetyl-GDEEKRNRAITARRQHLKSVMLQIAATELEKEEGRREAEK-NH2; TnI-(56–115), acetyl-SMAEVQELCKQLHAKIDAAEEEKYDMEIKVQKSSKELEDMNQKLFDLRGKFKRPPLRRVR-NH2; TnI-(96–139), acetyl-NQKLFDLRGKFKRPPLRRVRMSADAMLKALLGSKHKVCMDLRAN-NH2. Proteins and peptides were lyophilized and resuspended in NMR buffer or non-denaturing gel electrophoresis buffer in appropriate concentrations. All protein and peptide concen- trations were determined by amino acid analysis in duplicate. TnC complexes with TnT and TnI peptides were visualized on 10% glycerol-polyacrylamide gels (25Katayama E. Nozaki S. J. Biochem. (Tokyo). 1982; 91: 1449-1452Crossref PubMed Scopus (17) Google Scholar). Unlabeled recombinant TnC (or the individual domains) and the peptides were resuspended in 20 mm Hepes pH 7.6, 100 mm NaCl, and 5 mm CaCl2. TnT-(209–243) could not be resuspended in the buffer and was resuspended directly in TnC solution. Protein concentrations were determined to be 25 μm for TnC, 19 μm for CTnC, 16 μm for NTnC, 143 μm for TnT-(151–170), 104 μm for TnT-(160–193), 59 μm for TnT-(181–215), 128 μm for TnT-(209–243), 185 μm for TnT-(228–260), 89 μm for TnI-(1–40), 103 μm for TnI-(56–115), and 186 μm for TnI-(96–139). TnC and TnT or TnI peptides were mixed in ratios from 1:1 to 1:4. TnC/TnI/TnT mixtures were made in 1:1:1 ratios. The mixtures were incubated for at least 30 min at room temperature before analysis and then diluted in sample buffer and analyzed in 10% glycerol, 8% polyacrylamide gels as described (26Farah C.S. Miyamoto C.A. Ramos C.H.I. 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). Recombinant TnC uniformly labeled with15N and the peptide TnT-(160–193) were resuspended in 100 mm KCl, 10 mm imidazole, 20 mmdithiothreitol, 6 mm CaCl2, 0.03% sodium azide, and 0.2 mm 2,2-dimethyl-2-silapentane-5-sulfonic acid in 90% H2O, 10% D2O at pH 6.9. TnC concentration in the initial NMR sample was 0.6 mm, and TnT-(160–193) concentration in the stock solutions was 21 and 62 mm, respectively. Titration points of 0.14, 0.29, 0.43, 0.57, 0.71, 0.86, 1, 1.14, 1.29, 1.43, 1.87, and 2.3 molar equivalents of TnT-(160–193) were of the 21 mm were for the first 10 and of the 62 mm were for the last two and two-dimensional 1H,15N NMR were at titration Recombinant uniformly labeled with and and the peptide TnT-(160–193) were resuspended in 100 mm KCl, 10 mm imidazole, 20 mm 6 mm CaCl2, 0.03% sodium azide, and 0.2 mm 2,2-dimethyl-2-silapentane-5-sulfonic acid in 90% H2O, 10% D2O at pH 6.9. concentration in the initial NMR sample was mm, and TnT-(160–193) concentration in the stock was Titration points of and molar equivalents of TnT-(160–193) were of the stock were for and two-dimensional 1H,15N NMR were at titration A last was with mm and molar equivalents of TnT-(160–193). All and two-dimensional 1H,15N NMR were on a were using of or a of 10 and an of The 1H,15N NMR were using the by E. Kay and P. J. Chem. 1992; Scopus Google Scholar, O. Kay J.D. J. 1994; PubMed Scopus Google and were and respectively. were for titration The data were using the and the F. S. G. J. A. J. 1995; PubMed Scopus Google Scholar) and J. 1994; PubMed Scopus Google Scholar). The five TnT peptides in TnT-(160–193), TnT-(181–215), TnT-(209–243), and were mixed with TnC in the presence of Ca2+ and to a non-denaturing TnT-(160–193) and TnT-(228–260) are to the TnC binding to TnC TnT-(160–193) and TnT-(228–260) the in TnT responsible for the interaction with TnC. when TnT-(228–260) is mixed with TnC in ratios the TnC be in the gel not This results from the binding of of TnT-(228–260) to TnC. this this on the interactions between TnT-(160–193) and TnC. can be as a in the gel in the presence or of TnC of and not affect the TnC In the of Ca2+ mm 10 of the peptides TnC not between TnC protein or individual N or C domains) and TnT and TnI TnC was with or two peptides at a as described and and the was analyzed by non-denaturing gel of peptides proteins are at the of the and TnC in of the five TnT peptides was mixed with TnC. TnT-(160–193) and TnT-(228–260) the TnC and bind to TnC. The was for the peptides The was TnT-(160–193) mixed with the individual of TnC. TnT-(160–193) a of the but not the The was 1:4. TnT-(160–193) and TnI-(1–40) bind simultaneously to TnC, a of the TnC The in this gel was 1:1:1 TnT-(160–193) and compete for TnC. When both peptides are to TnC the is the by TnI-(96–139). The in this gel was 1:1:1 When the peptide TnT-(160–193) is mixed to the individual of TnC in the presence of Ca2+, a of the C domain but not of the N domain This that the interaction between TnT-(160–193) and TnC occurs the C domain of the The of the some binding to the N but for the complex is the of and TnT-(160–193), a complex be in the A complex while in the gel and not be as a TnI peptides described in TnI-(56–115), and were with TnC and TnT-(160–193) to how they affect the interaction between TnC and TnT-(160–193). TnT-(160–193) and the peptide containing the first 40 amino from TnI can bind to TnC When either of the two peptides is to TnC, the TnC is When both peptides are to TnC, the is to the of both individual This the binding of the two peptides to TnC. TnT-(160–193) binds to TnC with TnI-(1–40), a when mixed to TnC, TnI-(1–40) the When the two peptides are simultaneously to TnC, some of the is still of the binding of TnT-(160–193). When is with TnC and TnT-(160–193), a is The on the TnC is the by the binding of which binds to TnC with TnT-(160–193) and from The of can be from the of the TnC that TnT-(160–193) The was with TnI-(56–115) and TnT-(160–193). with TnI-(96–139), TnI-(56–115) also with TnT-(160–193), from binding TnC not TnT-(160–193) was and 13C,15NCTnC The binding was by NMR The two-dimensional 1H,15N NMR of 13C,15NCTnC in the and the of the titration are in and respectively. to and of the of an individual amino for some of the are in both The two are were the titration as a of the of the residues affected by TnT-(160–193). The binding occurs with fast on the NMR The in titration correspond to the of and This can be in which a of a region of the two-dimensional 1H,15N NMR of 13C,15NCTnC the While some do not for and are in as by the for and The when the 1, the binding of TnT-(160–193) of NMR titration with TnT-(160–193). A of the NMR of in the of TnT-(160–193) is in while in the region of the peptide are in Titration of TnC with TnT-(160–193) results for residues in the C-terminal in the N-terminal domain not the C-terminal domain was and then they to the N of two TnC can a TnC C.M. Kay C.M. Reinach F.C. Smillie L.B. Sykes B.D. Biochemistry. 1995; 34: 7365-7375Crossref PubMed Scopus (61) Google chemical shift changes in the N domain can be by of the chemical shift for residue were calculated from the chemical shift changes of both using R.T. Tripet B.P. Hodges R.S. Sykes B.D. J. Biol. Chem. 1997; 272: 28494-28500Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). A of the NMR chemical shift changes for residues in the C domain for both can be in The chemical shift changes for residue were in both for the first residues in This is by the for this region in the two While these residues are in the of the protein in TnC, in they are the N-terminal The residues in the C domain that a chemical shift 100 at the of the titration were and in both and the results were in the binding in for residues in the N domain that 40 were also to the C domain and in The were to binding using TnC both and The dissociation were 30 ± 6 μm for the C domain of TnC and ± μm for the N domain binding TnT-(160–193) concentrations were at for the TnT-(160–193) concentrations. The dissociation constant for the N domain be determined the site not the can be that is at least 10 for the C domain and that the binding to the N domain is not the C domain is 90% The for the N domain in 6 was the that the by the binding of TnT-(160–193) in were mapped on the protein surface both for the C domain in the presence of and for the TnC in the model of TnC·TnI The surface was in a to the to which the residues were affected by TnT-(160–193) in correspond to residues chemical to 1, not by 20 the were in this the site on TnC can be visualized in the protein and in the binary complex the five peptides the T2 region in rabbit fast skeletal muscle TnT, two peptides, containing residues 160–193 and 228–260, bind TnC to non-denaturing gel electrophoresis the in TnT. binding to be as a in the gel the is not to be results are in with results from the Ca2+-dependent binding to TnC of peptides from the T2 region or the T2 containing residues 160–193 J.R. Smillie L.B. J. Biochem. PubMed Scopus Google Scholar, J.R. Smillie L.B. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar, M. Y. A. J. Biochem. (Tokyo). PubMed Scopus Google Scholar, A. S.V. Biophys. 1989; PubMed Scopus Google Scholar). The smallest of residues is also of binding for peptides containing residues and J.R. Smillie L.B. J. Biochem. PubMed Scopus Google the last two peptides not complexes that could be by electrophoresis P. S.V. Biochem. J. PubMed Scopus Google Scholar). The peptide containing residues of TnT-(160–193), and with in these binding in the of Ca2+, in the presence of J.R. Smillie L.B. J. Biochem. PubMed Scopus Google Scholar, J.R. Smillie L.B. J. Biol. Chem. 1982; Full Text PDF PubMed Google Scholar, M. Y. A. J. Biochem. (Tokyo). PubMed Scopus Google Scholar). have that the C domain of TnC when to Ca2+ or G. K. L. Biochemistry. 1992; PubMed Scopus Google Scholar, A. P. J. Mol. 2000; Full Text PDF PubMed Scopus Google Scholar). TnT-(228–260) to have a primary binding site on TnC but also binds TnC in concentrations. This binding can affect and binding site NMR that the peptide when in and the complex in the NMR not these we this on the of TnT-(160–193) binding to TnC. The binding site for TnT-(160–193) is located on the C-terminal domain of TnC. is TnT-(160–193) capable of binding to CTnC, but the residues are in the binding of TnT-(160–193) to the protein and to CTnC, that the peptide is to the site in both When the C domain is TnT-(160–193) binds to the N domain of TnC. occurs with much 10 and is The dissociation constant for binding is 30 ± 6 μm. The between TnC and TnT was previously as C.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This can be by or but we a peptide the was made using the The presence of residues from TnT the The between the of TnT-(160–193) for and TnC is not the other the of the N-terminal residues of the binding to TnC. The titration not have points to a in the The binding data for the N domain in 6 has a the binding This could be the of of TnC. TnC the N domain C.M. Kay C.M. Reinach F.C. Smillie L.B. Sykes B.D. Biochemistry. 1995; 34: 7365-7375Crossref PubMed Scopus (61) Google and the chemical shift changes for the N domain are the effect of TnT-(160–193) binding and of the TnT-(160–193) binds to the N domain of TnC in the and both to the binding The site was mapped on the surface of TnC in of the two recently published for the TnC·TnI binary complex (15Tung C.-S. Wall M.E. Gallagher S.C. Trewhella J. Protein Sci. 2000; 9: 1312-1326Crossref PubMed Scopus (43) Google Scholar) The binding site and between the and in the of site the first binding of both and is also and residues to the C-terminal of the the binding site for TnT-(160–193). TnC in was to show the binding The surface not on the is and not affected by TnT-(160–193). shift changes in the N domain were not mapped on the protein surface they the of TnC. The region containing residues 160–193 in TnT in TnC binding is N-terminal from the of TnT a of a that has been to with residues of TnI, which also a J.R. Smillie L.B. J. Biochem. Scholar). A TnT containing residues of TnT bind to TnI J.R. Smillie L.B. J. Biochem. while a chicken TnT residues B. C.S. Reinach F.C. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and a TnT residues R. S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar) bind TnI. correspond to residues and in rabbit respectively. were also to be for TnI binding M. Y. A. J. Biochem. (Tokyo). PubMed Scopus Google Scholar). of TnT responsible for TnC and TnI binding are located by is that the of TnC and TnI that with TnT be located in the troponin complex. In the model in the region containing the in TnI (residues to the in to the of the C domain of which not with TnC in the complex. to the mapped site on TnC to that these could with in TnT. The non-denaturing gel electrophoresis data that TnT-(160–193) and TnI-(1–40) can bind to TnC at the The region of TnI to TnI-(1–40) is in both peptides bind to region of TnC, they do not with the residues and not compete for TnC NMR chemical shift changes mapping the site on that the binding the residues in the of (14Mercier P. Li M.X. Sykes B.D. Biochemistry. 2000; 39: 2902-2911Crossref PubMed Scopus (49) Google while TnT-(160–193) the surface of and in the region to the and TnI-(56–115) compete with TnT-(160–193) binding to TnC, that they at least of the binding site on TnC. the inhibitory peptide (residues as the to the of TnC in and an region (residues that binds to the N domain of TnC R.T. Tripet B.P. Hodges R.S. Sykes B.D. J. Biol. Chem. 1997; 272: 28494-28500Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The inhibitory peptide is the region between the two peptides, and be the region that with TnT-(160–193). The model for the TnC·TnI binary complex in not for that We that the binding site for the inhibitory peptide on TnC the at least The inhibitory peptide is located to TnT in the troponin complex, and the presence of TnT can the inhibitory region from Grabarek Z. J. Biophys. 1999; PubMed Scopus Google Scholar). for TnT-(160–193), has both and interactions with CTnC, while TnI-(1–40) has interactions (14Mercier P. Li M.X. Sykes B.D. Biochemistry. 2000; 39: 2902-2911Crossref PubMed Scopus (49) Google Scholar). TnT-(160–193) and some of the and compete for TnC. is to that while with TnI-(1–40) and with TnT-(160–193), TnI-(1–40) and TnT-(160–193) do not The binding site for the inhibitory peptide residues with and that the last two either do not or the region is that not affect the results much of model from (15Tung C.-S. Wall M.E. Gallagher S.C. Trewhella J. Protein Sci. 2000; 9: 1312-1326Crossref PubMed Scopus (43) Google for the of the inhibitory is not with The of TnI residues in model the mapped competition between TnI-(1–40) and TnT-(160–193). We that model is to the structure of the binary TnC·TnI complex. This is the first that a site has been mapped on TnC. When with the information available on TnI binding to TnC, the mapping how the three components in the structure of troponin complex. We for Corson for and TnC and and for of the NMR We and Spyracopoulos for and this