Identification of a Region of Fast Skeletal Troponin T Required for Stabilization of the Coiled-coil Formation with Troponin I

We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca2+-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the α helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform. We have previously identified evolutionarily conserved heptad hydrophobic repeat (HR) domains in all isoprotein members of troponin T (TnT) and troponin I (TnI), two subunits of the Ca2+-regulatory troponin complex. Our suggestion that the HR domains are involved in the formation of a coiled-coil heterodimer of TnT and TnI has been recently confirmed by the crystal structure of the core domain of the human cardiac troponin complex. Here we studied a series of recombinant deletion mutants of the fast skeletal TnT to determine the minimal sequence required for stable coiled-coil formation with the HR domain of the fast skeletal TnI. Using circular dichroism spectroscopy, we measured the α helical content of the coiled-coil formed by the various TnT peptides with TnI HR domain. Sedimentation equilibrium experiments confirmed that the individual peptides of TnT were monomeric but formed heterodimers when mixed with HR domain of TnI. Isothermal titration calorimetry was then used to directly measure the affinity of the TnT peptides for the TnI HR domain. Surprisingly we found that the HR regions alone of the fast skeletal TnT and TnI, as defined earlier, were insufficient to form a coiled-coil. Furthermore we showed that an additional 14 amino acid residues N-terminal to the conserved HR region (TnT residues 165-178) are essential for the stable coiled-coil formation. We discuss the implication of our finding in the fast skeletal troponin isoform in the light of the crystal structure of the cardiac isoform. Vertebrate striated muscle contraction is regulated by Ca2+, and the proteins that mediate this regulation in the contractile muscle are tropomyosin (Tm) 1The abbreviations used are: Tm, tropomyosin; Tn, troponin; TnT, troponin T; TnI, troponin I; TnC, troponin C; fsTnT, fast skeletal troponin T; fsTnI, fast skeletal troponin I; cTnT, cardiac TnT; HR, heptad hydrophobic repeat; TnT HR, peptide corresponding to the HR region of the fast skeletal troponin T; TnI HR, peptide corresponding to the HR region of the fast skeletal troponin I; TnT1, N-terminal proteolytic fragment of TnT; TnT2, C-terminal proteolytic fragment of TnT; ITC, isothermal titration calorimetry; βME, β-mercaptoethanol. and troponin (Tn) (for reviews, see Refs. 1Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1351) Google Scholar, 2Ebashi S. Endo M. Otsuki J. Q. Rev. Biophys. 1969; 2: 351-384Crossref PubMed Scopus (537) Google Scholar, 3Phillips Jr., G.N. Fillers J.P. Cohen C. J. Mol. Biol. 1986; 192: 111-131Crossref PubMed Scopus (270) Google Scholar, 4Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (476) Google Scholar, 5Leavis P.C. Gergely J. CRC Crit. Rev. Biochem. 1984; 16: 235-305Crossref PubMed Scopus (326) Google Scholar, 6Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-539Crossref PubMed Scopus (450) Google Scholar). The Tm-Tn complex binds polymerized actin to form the regulated thin filament. Actin, Tm, and Tn are present in the thin filament in a ratio of 7:1:1 (2Ebashi S. Endo M. Otsuki J. Q. Rev. Biophys. 1969; 2: 351-384Crossref PubMed Scopus (537) Google Scholar). Tm is an α helical coiled-coil protein and interacts with another Tm molecule in a head-to-tail manner to form a strand that lies in the groove of the polymerized actin filament (2Ebashi S. Endo M. Otsuki J. Q. Rev. Biophys. 1969; 2: 351-384Crossref PubMed Scopus (537) Google Scholar, 3Phillips Jr., G.N. Fillers J.P. Cohen C. J. Mol. Biol. 1986; 192: 111-131Crossref PubMed Scopus (270) Google Scholar). The Tn complex is composed of three structurally and functionally different proteins: troponin C (TnC), which binds to Ca2+; troponin I (TnI), which binds to actin; and troponin T (TnT), which binds to Tm. In the relaxed muscle, TnI inhibits the ATPase activity of actomyosin by binding to actin and blocking the actin-myosin interaction (1Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1351) Google Scholar, 4Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (476) Google Scholar, 5Leavis P.C. Gergely J. CRC Crit. Rev. Biochem. 1984; 16: 235-305Crossref PubMed Scopus (326) Google Scholar, 6Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-539Crossref PubMed Scopus (450) Google Scholar). In the excited muscle, depolarization of muscle membrane releases Ca2+ in the sarcoplasm, and the binding of this Ca2+ to TnC leads to a conformational change in TnC. This initiates muscle contraction through a process of “information transfer.” Subsequent steps involve multiprotein interactions and conformational changes in the thin filament leading to the contraction of muscle. The information transfer during muscle contraction presumably follows the order: TnC → TnI → TnT → Tm → actin (1Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1351) Google Scholar, 4Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (476) Google Scholar, 5Leavis P.C. Gergely J. CRC Crit. Rev. Biochem. 1984; 16: 235-305Crossref PubMed Scopus (326) Google Scholar, 6Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-539Crossref PubMed Scopus (450) Google Scholar). All of the subunits of the Tn complex participate in binary and ternary interactions, and these play a critical role in the Ca2+ regulation of the muscle contraction. The natures of these interactions as well as the factors that influence them are key for understanding the contraction mechanism (1Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1351) Google Scholar, 4Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (476) Google Scholar, 5Leavis P.C. Gergely J. CRC Crit. Rev. Biochem. 1984; 16: 235-305Crossref PubMed Scopus (326) Google Scholar, 6Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-539Crossref PubMed Scopus (450) Google Scholar). The involvement of TnT is particularly important in the regulatory system as TnT interacts with TnI, TnC, Tm, and actin (7Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (259) Google Scholar). TnT anchors the whole Tn complex to the thin filament by interacting with the Tm head-to-tail overlapping region and thus plays a major structural role (3Phillips Jr., G.N. Fillers J.P. Cohen C. J. Mol. Biol. 1986; 192: 111-131Crossref PubMed Scopus (270) Google Scholar, 4Farah C.S. Reinach F.C. FASEB J. 1995; 9: 755-767Crossref PubMed Scopus (476) Google Scholar, 7Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (259) Google Scholar). Another of TnT is to the of the is required for the in the ATPase to the of Ca2+ S. 1995; PubMed Scopus Google Scholar, J. Mol. Biol. 1998; PubMed Scopus Google Scholar, Biophys. J. PubMed Scopus Google Scholar). in the for the regions of TnT involved in interactions and important for have used TnT recombinant (7Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (259) Google Scholar, J.D. J. J. Biol. Chem. 1995; PubMed Scopus Google Scholar, C.S. Reinach F.C. J. Biol. Chem. 1998; PubMed Scopus Google Scholar, P.C. S. PubMed Scopus Google Scholar, S. 1998; PubMed Scopus Google Scholar, C.S. Reinach F.C. J. Biol. Chem. 2000; PubMed Scopus Google Scholar). skeletal TnT by proteolytic two and (7Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (259) Google Scholar). of the and the of interactions of TnT with thin filament proteins are these two (7Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (259) Google Scholar, S. 1995; PubMed Scopus Google Scholar, J.D. S. PubMed Scopus Google Scholar). The fragment is involved in the interaction with Tm, the which is in the regulatory of the Tn is involved in the interactions with TnC and TnI (7Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (259) Google Scholar). TnT residues have been to with TnC (7Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (259) Google Scholar, J. Biol. Chem. PubMed Scopus Google and residues have been as the TnI interaction region (7Perry S.V. J. Muscle Res. Cell Motil. 1998; 19: 575-602Crossref PubMed Scopus (259) Google Scholar, C.S. Reinach F.C. J. Biol. Chem. 1998; PubMed Scopus Google Scholar). This region an evolutionarily conserved region previously identified by with heptad hydrophobic for to form coiled-coil S. S. 1998; PubMed Scopus Google Scholar, J. Biochem. Scholar). presumably a is present in TnI S.V. Mol. Biochem. PubMed Google Scholar). and deletion of the HR region of and of interaction with the was that these regions of TnT and TnI form a α helical coiled-coil S. S. 1998; PubMed Scopus Google Scholar). of the crystal structure of the core domain of the human cardiac Tn cardiac TnC and the C-terminal of cardiac TnI and TnT, confirmed these interactions S. PubMed Scopus Google Scholar). In the crystal residues of an α helical coiled-coil with residues of The of the coiled-coil region as a major structural domain in the Tn complex is the crystal structure of the cardiac Tn core complex. an understanding of the particularly this region in the fast skeletal isoform plays a role in the binary interaction with TnI, is to of crystal structure of the fast skeletal isoform. the in the present we the of the coiled-coil formation of the fast skeletal Tn isoform recombinant peptides TnT and TnI. We the region amino acid residues and to form a coiled-coil with TnI We showed that a TnT fragment the HR region a coiled-coil with the HR region of TnI, a TnT fragment the additional amino acid residues N-terminal to the HR a coiled-coil. was that a region the HR domain of is required for the of this and were were and were were The and used for protein were of for of for the of the and deletion mutants were the human fast skeletal troponin T sequence P.C. S. Cell Biol. 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This ratio is for a stable coiled-coil PubMed Scopus Google Scholar, J. Biol. Chem. 1984; PubMed Google Scholar, J. Biol. Chem. PubMed Google Scholar, M. J. J. 1998; PubMed Scopus Google Scholar). the HR regions of cardiac TnT and TnI form a coiled-coil S. 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S. 1998; PubMed Scopus Google that the HR domains are involved in a coiled-coil formation. This is with the recently crystal structure of the human cardiac Tn core domain that the of a coiled-coil structure a acid sequence of the HR domains of and S. PubMed Scopus Google Scholar). structural information is for the fast skeletal we used recombinant of and to determine the minimal sequence of TnT required for the formation of a stable coiled-coil with TnI Our that and deletion as well as TnI HR are but the of α are and are by the of In of different and TnI HR showed in α helical content a of the structure when the with as in the of a coiled-coil Sedimentation equilibrium that of TnI HR and different formed were all We these that TnI HR and with through the formation of a α helical coiled-coil. all the with TnI HR to the The of amino acid residues in the was required for the formation and of the coiled-coil. (TnT residues and formed stable coiled-coil with TnI This is by our that the for the interaction with TnI HR was for the the residues the them The that the of the coiled-coil formed by was the coiled-coil formed by that the of residues additional to the coiled-coil the TnT residues to have the interaction as the coiled-coil formed by and TnT HR the of the recently crystal structure of the cardiac Tn core domain S. PubMed Scopus Google and the C-terminal fragment of the of the core domain of Tn to Tm through for and residues for is to have an α helical which binds with Tm through hydrophobic the coiled-coil and the the domain to the core domain. The of this region with the of the TnT molecule was well defined in the crystal to this residues of cTnT, the coiled-coil form an α that is a to the coiled-coil. is that the corresponding amino acid residues in in the as that of the cardiac isoform. Our that residues of are required for formation of a stable coiled-coil with TnI HR, this region is the coiled-coil of our is that this region acid residues is to the coiled-coil domain is in the crystal structure and thus the coiled-coil structure by directly interacting with We have previously that a TnT with TnI when was to S. 1998; PubMed Scopus Google Scholar). The binary was in the of Ca2+ and in the ternary complex in the of Ca2+ that the region amino acid was to TnI to the formation of a the interaction that that residues interaction with TnI that them C.S. J. Biol. Chem. PubMed Google Scholar, J. Biochem. Scholar). Furthermore of the TnT HR sequence by coiled-coil as that the sequence a of coiled-coil formation with S. S. 1998; PubMed Scopus Google Scholar). the cardiac coiled-coil for regions required to the coiled-coil the coiled-coil domains of cardiac and fast skeletal this these our that the region of residues plays an important role in the of the the in the the of the region of amino acid residues and and in the light of the crystal structure of the Tn core domain of the cardiac we a the of this region to the coiled-coil structure in the fast skeletal complex. of the TnT fragment residues and and the fragment of TnT TnI in the coiled-coil structure the coiled-coil presumably through The TnT region interacts with the amino acid residues of the TnI HR the two amino acid and of TnT participate in interactions with residues and the TnI HR fragment the a that is present in the cTnT, and this to in the of the region cardiac and fast skeletal with the that a of amino acid residues are present the region this the of additional The cardiac crystal structure the of the fragment involved in the coiled-coil interaction as residues S. PubMed Scopus Google Scholar). is that for the skeletal Tn isoform the of the TnT involved in the coiled-coil structure the Our showed that deletion of the C-terminal of the HR region and the coiled-coil This that the C-terminal of the coiled-coil are the for cardiac and fast skeletal as in to of the the 14 amino acid residues the of the conserved HR region of with the residues the fragment required for the of the coiled-coil in the fast skeletal isoform. In of coiled-coil hydrophobic residues and of the heptad residues the through interactions PubMed Scopus Google Scholar, C. J. PubMed Scopus Google Scholar, M. J. Biol. PubMed Scopus Google Scholar). Our that the TnT region the coiled-coil domain plays a role in the coiled-coil This a of interactions residues in the region formation of a structure the of the fast skeletal coiled-coil. of this region is the conserved of the amino acid of TnT different to human and different muscle S. PubMed Scopus Google conserved amino acid acid and In the region of TnT the coiled-coil region conserved The of sequence in the region is and that important for the structural of the Tn as of these residues are for formation. Our the cardiac the of the of the is of with the conserved amino acid to conformational the cardiac and fast skeletal We are to our and to the of the conserved amino acid residues in the fast skeletal isoform by the of a series of