Structural Consequences of Cardiac Troponin I Phosphorylation

β-Adrenergic stimulation of the heart results in bisphosphorylation of the N-terminal extension of cardiac troponin I (TnI). Bisphosphorylation of TnI reduces the affinity of the regulatory site on troponin C (TnC) for Ca2+ by increasing the rate of Ca2+ dissociation. What remains unclear is how the phosphorylation signal is transmitted from one subunit of troponin to another. We have produced a series of mutations in the N-terminal extension of TnI designed to further our understanding of the mechanisms involved. The ability of phosphorylation of the mutant TnIs to affect Ca2+ sensitivity has been assessed. We find that the Pro residues found in a conserved (Xaa-Pro)4 motif N-terminal to the phosphorylation sites are not required for the effect of the N-terminal extension on Ca2+ binding in the presence or absence of phosphorylation. Our experiments also reveal that the full effects of phosphorylation are seen even when residues 1–15 of TnI are deleted. If further residues are removed, not only does the effect of phosphorylation diminish but deletion of the N-terminal extension mimics phosphorylation. We propose that TnI residues 16–29 bind to TnC stabilizing the “open” Ca2+-bound state. Phosphorylation (or deletion) prevents this binding, accelerating Ca2+ release. β-Adrenergic stimulation of the heart results in bisphosphorylation of the N-terminal extension of cardiac troponin I (TnI). Bisphosphorylation of TnI reduces the affinity of the regulatory site on troponin C (TnC) for Ca2+ by increasing the rate of Ca2+ dissociation. What remains unclear is how the phosphorylation signal is transmitted from one subunit of troponin to another. We have produced a series of mutations in the N-terminal extension of TnI designed to further our understanding of the mechanisms involved. The ability of phosphorylation of the mutant TnIs to affect Ca2+ sensitivity has been assessed. We find that the Pro residues found in a conserved (Xaa-Pro)4 motif N-terminal to the phosphorylation sites are not required for the effect of the N-terminal extension on Ca2+ binding in the presence or absence of phosphorylation. Our experiments also reveal that the full effects of phosphorylation are seen even when residues 1–15 of TnI are deleted. If further residues are removed, not only does the effect of phosphorylation diminish but deletion of the N-terminal extension mimics phosphorylation. We propose that TnI residues 16–29 bind to TnC stabilizing the “open” Ca2+-bound state. Phosphorylation (or deletion) prevents this binding, accelerating Ca2+ release. troponin C troponin I troponin T cAMP-dependent protein kinase (EC 2.7.1.37) 2-(4′-(iodoacetamido)anilino)napthalene-6-sulfonic acid 4-morpholinepropanesulfonic acid Troponin, working in conjunction with tropomyosin, acts as a molecular switch, regulating muscle contraction in response to changes in the intracellular Ca2+ concentration. Troponin is a complex of three proteins: the Ca2+ binding subunit, troponin C (TnC),1 the inhibitory subunit, troponin I (TnI), and the tropomyosin binding subunit, troponin T (TnT). Crystal and NMR structures (1Herzberg O. James M.N.G. Nature. 1985; 313: 653-659Crossref PubMed Scopus (479) Google Scholar, 2Sia S.K., Li, M.X. Spyracopoulos L. Gagne S.M. Liu W. Putkey J.A. Sykes B.D. J. Biol. Chem. 1997; 272: 18216-18221Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) reveal that TnC is a dumbbell-shaped molecule, consisting of a C-terminal domain possessing two Ca2+/Mg2+ binding sites (sites III and IV) and a N-terminal domain possessing two Ca2+-specific binding sites (sites I and II) in the skeletal isoform. The binding of Ca2+ at sites I and II produces a structural opening of the N-terminal domain exposing additional TnI binding sites (1Herzberg O. James M.N.G. Nature. 1985; 313: 653-659Crossref PubMed Scopus (479) Google Scholar, 3Houdusse A. Love M.L. Dominguez R. Grabarek Z. Cohen C. Structure. 1997; 5: 1695-1711Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). In cardiac TnC site I is inactive (4Van Eerd J.P. Takahashi K. Biochemistry. 1976; 15: 1171-1180Crossref PubMed Scopus (184) Google Scholar), and it is Ca2+ binding to site II that is the primary regulator of cardiac contractility. Binding of Ca2+to the single “regulatory” site of cardiac TnC alone is not sufficient to stabilize the open conformation of the N-terminal domain in the absence of TnI (2Sia S.K., Li, M.X. Spyracopoulos L. Gagne S.M. Liu W. Putkey J.A. Sykes B.D. J. Biol. Chem. 1997; 272: 18216-18221Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). However, in the presence of TnI and Ca2+ the N-terminal domain of cardiac TnC also adopts the open conformation (5Li M.X. Spyracopoulos L. Sykes B.D. Biochemistry. 1999; 38: 8289-8298Crossref PubMed Scopus (243) Google Scholar). Although we do not yet have atomic resolution structures of TnI and TnT, much information has been afforded by studies with fragments and mutants of these proteins. TnI is an extended molecule that adopts an antiparallel orientation to TnC in the binary complex (6Dong W.-J. Xing J. Solaro J.R. Cheung H.C. Proteins Struct. Funct. Genet. 2000; 41: 438-447Crossref PubMed Scopus (24) Google Scholar, 7Farah C.S. Miyamoto C.A. Ramos C.H.I. da Silva A.C.R. Quaggio R.B. Fujimora K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar). Cardiac TnI (209 amino acids) has a 31-residue extension not present in other isoforms. Fig. 1 shows the aligned sequences of the N-terminal extensions of the mammalian cardiac TnIs sequenced to date (8Ausoni S. Campione M. Picard A. Moretti P. Vitadello M., De Nardi C. Schiaffino S. J. Biol. Chem. 1994; 269: 339-364Abstract Full Text PDF PubMed Google Scholar, 9Grand R.J.A. Wilkinson J.M. Mole L.E. Biochem. J. 1976; 159: 633-641Crossref PubMed Scopus (61) Google Scholar, 10Leszyk J. Dumaswala R. Potter J.D. Collins J.H. Biochemistry. 1988; 27: 2821-2827Crossref PubMed Scopus (71) Google Scholar, 11Murphy A.M. Jones L. Sims H.F. Strauss A.W. Biochemistry. 1991; 30: 707-712Crossref PubMed Scopus (98) Google Scholar, 12Vallins W.J. Brand N.J. Dabhade N. Butler-Browne G. Yacoub M.H. Barton P.J.R. FEBS Lett. 1990; 270: 57-61Crossref PubMed Scopus (127) Google Scholar). Within this extension are two residues, serines 22 and 23, which are substrates for cAMP-dependent protein kinase (PKA). C-terminal to the extension, residues 33–80 of cardiac TnI form an α-helix that binds extremely tightly to the C-terminal domain of TnC (13Gasmi-Seabrook G. Howarth J.W. Finley N. Abusamhadneh E. Gaponenko V. Brito R.M.M. Solaro J.R. Rosevear P.R. Biochemistry. 1999; 38: 8313-8322Crossref PubMed Scopus (48) Google Scholar,14Vassylyev D.G. Takeda S. Wakatsuki S. Maeda K. Maeda Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4847-4852Crossref PubMed Scopus (191) Google Scholar). 2D. G. Ward and I. P. Trayer, unpublished data. 2D. G. Ward and I. P. Trayer, unpublished data. This is followed by a putative TnT binding site and then the “inhibitory” (115–131) and “regulatory” (147–163) regions (5Li M.X. Spyracopoulos L. Sykes B.D. Biochemistry. 1999; 38: 8289-8298Crossref PubMed Scopus (243) Google Scholar, 15McKay R.T. Pearlstone J.R. Corson D.C. Gagne S.M. Smillie L.B. Sykes B.D. Biochemistry. 1998; 37: 12419-12430Crossref PubMed Scopus (49) Google Scholar). These latter regions bind to Ca2+-saturated TnC but, in the absence of Ca2+, bind to actin-anchoring troponin and tropomyosin in the “blocked state” of the McKillop and Geeves (16McKillop D.F. Geeves M.A. Biophys. J. 1993; 65: 693-701Abstract Full Text PDF PubMed Scopus (649) Google Scholar) model of thin filament regulation. Serines 22 and 23 of cardiac TnI are phosphorylated by PKA in response to β-adrenergic stimulation of the heart. This bisphosphorylation desensitizes the thin filaments to Ca2+ by 0.2–0.4pCa units (17Dohet C., Al- Hillawi E. Trayer I.P. Ruegg J.C. FEBS Lett. 1995; 377: 131-134Crossref PubMed Scopus (40) Google Scholar, 18Ray K.P. England P.J. FEBS Lett. 1976; 70: 11-16Crossref PubMed Scopus (132) Google Scholar, 19Robertson S.P. Johnson D.J. Holroyde M.J. Kranias E.G. Potter J.D. Solaro J.R. J. Biol. Chem. 1982; 257: 260-263Abstract Full Text PDF PubMed Google Scholar) due to an increase in the rate of Ca2+ dissociation from the N-terminal regulatory site on TnC (19Robertson S.P. Johnson D.J. Holroyde M.J. Kranias E.G. Potter J.D. Solaro J.R. J. Biol. Chem. 1982; 257: 260-263Abstract Full Text PDF PubMed Google Scholar). The faster release of Ca2+ produces faster relaxation of skinned fibers upon rapid Ca2+ chelation (20Zhang R. Zhao J.J. Mandveno A. Potter J.D. Circ. Res. 1995; 76: 1028-1035Crossref PubMed Scopus (267) Google Scholar). This enhanced relaxation, in conjunction with increased Ca2+ re-uptake into the sarcoplasmic reticulum, may help maintain proper diastolic function at increased heart rates. Despite intensive efforts, it remains unclear how the phosphorylation signal is transmitted from the N-terminal extension of TnI to the regulatory Ca2+ binding site in the N-terminal domain of TnC, especially in view of the antiparallel orientation of the two proteins. 1H NMR experiments on peptides corresponding to regions of the N-terminal extension show that the unphosphorylated extension is highly flexible but that bisphosphorylation increases the number of conformational restraints with residues 24–29 adopting a looped conformation (21Keane N.E. Quirke P.G. Gao Y. Patchell V.B. Perry S.V. Levine B.A. Eur. J. Biochem. 1997; 248: 329-337Crossref PubMed Scopus (31) Google Scholar, 22Jaquet K. Lohmann K. Czisch M. Holak T. Gulati J. Jaquet R. J. Muscle Res. Cell Motil. 1998; 19: 647-659Crossref PubMed Scopus (11) Google Scholar). 1H and 31P NMR on these peptides or whole TnI indicates that phosphoserine 23 interacts with Arg21, whereas phosphoserine 22 behaves as if free in solution (22Jaquet K. Lohmann K. Czisch M. Holak T. Gulati J. Jaquet R. J. Muscle Res. Cell Motil. 1998; 19: 647-659Crossref PubMed Scopus (11) Google Scholar, 23Jaquet K. Korte K. Schnackerz K. Vyska K. Heilmeyer L.M.G. Biochemistry. 1993; 32: 13873-13878Crossref PubMed Scopus (25) Google Scholar). In bisphosphorylated troponin the phosphate groups are in a more acidic environment than in isolated TnI, which suggests that the phosphorylated extension may interact weakly with another component of troponin. However, the narrow31P line widths observed were interpreted to indicate that the phosphate groups themselves are not involved in specific bond formation (23Jaquet K. Korte K. Schnackerz K. Vyska K. Heilmeyer L.M.G. Biochemistry. 1993; 32: 13873-13878Crossref PubMed Scopus (25) Google Scholar). Isolated TnI and the TnI·TnC complex both adopt a more compact shape upon phosphorylation (24Liao R. Wang Cheung H.C. Biophys. J. Full Text PDF PubMed Scopus Google Scholar) and experiments have that the N-terminal of a mutant TnI with 2-(4′-(iodoacetamido)anilino)napthalene-6-sulfonic acid the of TnI W.-J. M. Xing J. M. Solaro J.R. Cheung H.C. Biochemistry. 1997; PubMed Scopus Google Scholar). Binding experiments peptides corresponding to the N-terminal extension have that the unphosphorylated form bind to TnC, whereas the phosphorylated form does not (21Keane N.E. Quirke P.G. Gao Y. Patchell V.B. Perry S.V. Levine B.A. Eur. J. Biochem. 1997; 248: 329-337Crossref PubMed Scopus (31) Google Scholar, G. M. J.C. S. M. P. C. FEBS Lett. 2000; PubMed Scopus Google Scholar, M.J. Trayer I.P. J. Muscle Res. Cell Motil. 1999; Scholar). The is by NMR and which indicate that the unphosphorylated N-terminal extension the N-terminal domain of TnC in both TnI·TnC binary complex and in whole troponin W.-J. A. Cheung H.C. Rosevear P.R. Biochemistry. PubMed Scopus Google Scholar, N. Abusamhadneh E. Gaponenko V. W.-J. G. Howarth J.W. M. Solaro J.R. Cheung H.C. Rosevear P.R. FEBS Lett. 1999; PubMed Scopus Google Scholar, V. Abusamhadneh E. Finley N. G. Solaro J.R. M. Rosevear P.R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, D.G. Trayer I.P. J. Muscle Res. Cell Motil. 2000; Scholar). Bisphosphorylation results in a in the on the N-terminal extension from to and it is that this the of the of serines 22 and 23 with residues mimics the effects of phosphorylation in skinned fibers (17Dohet C., Al- Hillawi E. Trayer I.P. Ruegg J.C. FEBS Lett. 1995; 377: 131-134Crossref PubMed Scopus (40) Google Scholar) but does not the conformational in a TnI as phosphorylation (21Keane N.E. Quirke P.G. Gao Y. Patchell V.B. Perry S.V. Levine B.A. Eur. J. Biochem. 1997; 248: 329-337Crossref PubMed Scopus (31) Google Scholar). that the phosphorylation signal is transmitted from TnI to TnC by in a the N-terminal extension and the N-terminal domain of We the structural of the N-terminal extension required for of the phosphorylation has been that sequences an extended conformation of the Levine B.A. Trayer I.P. Eur. J. Biochem. PubMed Scopus Google Scholar, S. M. Trayer I.P. G. Levine B.A. J. Biol. 1990; PubMed Scopus Google Scholar). 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Scholar). protein were the acid protein with were produced by of and at the of and and PKA from from from from other were from England were from by with an site and with site as E. Trayer I.P. Eur. J. Biochem. 1994; PubMed Scopus Google Scholar). were by by followed by a to the to the whole The N-terminal of TnI were a single the were into and into E. were for by and Proteins were in E. and by in 1 1 as E. Trayer I.P. Eur. J. Biochem. 1994; PubMed Scopus Google Scholar). and were by and D.G. P.R. Trayer Trayer I.P. Eur. J. Biochem. PubMed Scopus Google Scholar). The troponin by TnC, TnI, and TnT in a in followed by 1 PKA also 1 tropomyosin, 1 1 and to the to of the to the the at for with or units of In experiments the PKA were and in of from the a at The to from M. Eur. J. Biochem. PubMed Scopus Google the were by the of of This followed by of and of The at at experiments that the to the that release our and that the of not mutant TnC at with by in 1 and at with a of by The TnC then into troponin by with TnI and TnT in a in The complex and then to and 1 PKA also The troponin for 1 at with or units the troponin to 1 in to The rate of Ca2+ release from the of upon Ca2+ from the regulatory site W.-J. Wang A.M. Cheung H.C. Biophys. J. 1997; Full Text PDF PubMed Scopus Google Scholar) by rapid with an of This by of 1 troponin with of at with a at were by of the to a single shows a The of in our experiments were and of at and The to the from which we the The from at of the were for unphosphorylated troponin and for bisphosphorylated phosphorylation reduces the by units This is the in the (17Dohet C., Al- Hillawi E. Trayer I.P. Ruegg J.C. FEBS Lett. 1995; 377: 131-134Crossref PubMed Scopus (40) Google Scholar, 18Ray K.P. England P.J. FEBS Lett. 1976; 70: 11-16Crossref PubMed Scopus (132) Google Scholar, 19Robertson S.P. Johnson D.J. Holroyde M.J. Kranias E.G. Potter J.D. Solaro J.R. J. Biol. Chem. 1982; 257: 260-263Abstract Full Text PDF PubMed Google Scholar). The and and the of were not by phosphorylation or by of the mutations in this experiments not to the thin filaments followed by and that the TnI phosphorylated increase in phosphorylation with and that other thin filament were The of Ca2+ release from unphosphorylated and phosphorylated troponin TnI and TnT and is in In both the is by a single Ca2+ release is from the to increase in the of the to inactive site I of TnC produced by of site The rate were for unphosphorylated troponin and for phosphorylated troponin. This increase in the dissociation rate by TnI phosphorylation is than the of in by (19Robertson S.P. Johnson D.J. Holroyde M.J. Kranias E.G. Potter J.D. Solaro J.R. J. Biol. Chem. 1982; 257: 260-263Abstract Full Text PDF PubMed Google Scholar) cardiac troponin but is to and We produced single and Pro mutants of The if this as a then of the Pro residues with increase and of the phosphorylation with three mutants phosphate to the as TnI not The results of the and experiments are in and three mutants much TnI with to the effects of with PKA produces a to the in the and an increase in the Ca2+ dissociation rate These results indicate that of the Pro residues is and that the of the is not required to the phosphorylation signal to and Pro TnI Pro TnI effects of the Pro mutations of TnI and phosphorylation on the Ca2+ sensitivity of and Ca2+ dissociation rate In both the are the of in a The effects of the Pro mutations of TnI and phosphorylation on the Ca2+ sensitivity of and Ca2+ dissociation rate In both the are the of We a series of TnI mutants with an increasing number of residues from the and The to were designed to the of the of TnI N-terminal to the phosphorylation of the mutants to and were phosphorylated by PKA at the rate and to the as TnI The mutant is not phosphorylated by the motif for PKA has been Fig. and in the mutant the phosphorylation sites are deleted. The and mutants were designed as a that the observed effects were due to phosphorylation of serines 22 and 23 and the effect of the whole of the N-terminal The in the experiments with the N-terminal deletion mutants of TnI are in II and the number of residues in Fig. The upon phosphorylation is for the deletion mutants to and This shows that residues 1–15 only a in the of the phosphorylation C-terminal to the ability of phosphorylation to a to Ca2+ an for residues one PKA has effect on the and However, as we residues C-terminal to we a to Ca2+ in the unphosphorylated that mimics the effect of phosphorylation. it that residues 16–29 of TnI are for cardiac troponin in the Ca2+ affinity unphosphorylated N-terminal of results with N-terminal deletion mutants of The are the of at in a results with N-terminal deletion mutants of The are the of at of Ca2+ release to the effects of phosphorylation on the deletion The dissociation rate are in and are the number of residues in Fig. The in the seen in the to residues from the of TnI the ability of phosphorylation to Ca2+ release. We find that the effect of phosphorylation on the Ca2+ The effect of the N-terminal extension is to Ca2+ release the effects of phosphorylation as seen in the In the N-terminal extension is more at Ca2+ binding than phosphorylation. These results show that it is the unphosphorylated form of the N-terminal extension that Ca2+ binding and not the phosphorylated form that Ca2+ N-terminal of dissociation from troponin with TnI deletion The are the of at in a Ca2+ dissociation from troponin with TnI deletion The are the of at shows the in experiments the troponin skeletal TnC or TnT than three cardiac the cardiac TnT with skeletal TnT not the ability of phosphorylation to a TnT also to the of Ca2+ release not These results indicate that the of cardiac TnT with to skeletal TnT are not to the phosphorylation However, when the cardiac TnC with the skeletal phosphorylation of the TnI to Ca2+ This may due to a specific skeletal TnC not a binding site for the N-terminal extension, or the of the skeletal TnC N-terminal domain the binding of two Ca2+ than the effects of results with troponin in which the cardiac TnT or TnC have been with the skeletal isoforms. The are the of in a results with troponin in which the cardiac TnT or TnC have been with the skeletal isoforms. The are the of In our experiments we effects of bisphosphorylation of cardiac TnI by PKA as (17Dohet C., Al- Hillawi E. Trayer I.P. Ruegg J.C. FEBS Lett. 1995; 377: 131-134Crossref PubMed Scopus (40) Google Scholar, 18Ray K.P. England P.J. FEBS Lett. 1976; 70: 11-16Crossref PubMed Scopus (132) Google Scholar, 19Robertson S.P. Johnson D.J. Holroyde M.J. Kranias E.G. Potter J.D. Solaro J.R. J. Biol. Chem. 1982; 257: 260-263Abstract Full Text PDF PubMed Google Scholar). Bisphosphorylation increases the rate from troponin by and desensitizes Ca2+ by We have these effects to the ability of mutations in the N-terminal extension of TnI to the of the phosphorylation signal to the regulatory Ca2+ site in the N-terminal domain of The of the N-terminal extension to that the conformational changes produced in the of the phosphoserine residues conformation (21Keane N.E. Quirke P.G. Gao Y. Patchell V.B. Perry S.V. Levine B.A. Eur. J. Biochem. 1997; 248: 329-337Crossref PubMed Scopus (31) Google Scholar, 22Jaquet K. Lohmann K. Czisch M. Holak T. Gulati J. Jaquet R. J. Muscle Res. Cell Motil. 1998; 19: 647-659Crossref PubMed Scopus (11) Google Scholar) transmitted a to a TnC binding site in the residues of We that this is not the The Pro residues in the putative to the effects of phosphorylation. residues 1–15 of TnI with in the effects of to that the N-terminal of TnI is not required to the phosphorylation signal to to cardiac TnI residues are present in mammalian cardiac TnIs sequenced to date but are conserved than the of TnI and may a other than other than the phosphorylation In and M. J. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) found that of residues of muscle regulatory the effects of phosphorylation of In TnI, of residues N-terminal to the phosphorylation sites only reduces but does not the effect of the effects of phosphorylation are in the and This suggests that and may involved in the we that of to to The phosphorylation of serines 22 and 23 into a of the N-terminal We to the effects of phosphorylation and were that these residues to the N-terminal of the bisphosphorylation motif were of much to TnC binding or a structural in the unphosphorylated or bisphosphorylated conformation of the N-terminal extension of in this is that residues 16–29 mimics phosphorylation. This that it is the unphosphorylated form of the N-terminal extension that is in stabilizing Ca2+ binding at the regulatory This is the of that by J. Solaro J. 1995; 27: Full Text PDF PubMed Scopus Google Scholar) in a of cardiac TnI with cardiac TnI with residues deleted. In skinned experiments the and TnI the and that the phosphorylated extension is the form that the Ca2+-bound form of However, of the bisphosphorylated the two studies on skinned fibers and one with in which the to the by phosphorylation J. A.M. Solaro J.R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, V. Solaro J.R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, M. J.J. Solaro J.R. Biochem. Biophys. Res. 1999; PubMed Scopus Google Scholar). binding studies show that peptides corresponding to the N-terminal extension bind to TnC with an affinity of in the unphosphorylated and that phosphorylation this binding G. M. J.C. S. M. P. C. FEBS Lett. 2000; PubMed Scopus Google Scholar, M.J. Trayer I.P. J. Muscle Res. Cell Motil. 1999; Scholar). of these studies the TnC binding site on the N-terminal extension as residues and and and These are the residues when removed, phosphorylation. the that the affinity of whole TnI for TnC is upon phosphorylation may from the of the N-terminal binding R. Wang Cheung H.C. Biochemistry. 1994; PubMed Scopus Google Scholar). line of is that of NMR studies by Rosevear and W.-J. A. Cheung H.C. Rosevear P.R. Biochemistry. PubMed Scopus Google Scholar, N. Abusamhadneh E. Gaponenko V. W.-J. G. Howarth J.W. M. Solaro J.R. Cheung H.C. Rosevear P.R. FEBS Lett. 1999; PubMed Scopus Google Scholar, V. Abusamhadneh E. Finley N. G. Solaro J.R. M. Rosevear P.R. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). These experiments show that a of cardiac TnI the TnC C-terminal domain binding site residues and the N-terminal extension, with the N-terminal domain of TnC in the inactive Ca2+ binding site These are not seen if serines 22 and 23 are to if the is or if the is with if the N-terminal extension is deleted. also in phosphorylation of TnI the the open and of the N-terminal domain of TnC and deletion of residues phosphorylation. We propose that when TnI is unphosphorylated residues of TnI bind to the N-terminal domain of TnC stabilizing the “open” Ca2+-bound phosphorylation of serines 22 and 23, the of and conformational changes the N-terminal extension of TnI reduces affinity for TnC the of the open conformation and Ca2+ release. 1–15 are not in this but of the TnC binding has the effect as phosphorylation. is required to the residues 16–29 of TnI and of serines 22 and 23 with has effect on Ca2+ sensitivity (17Dohet C., Al- Hillawi E. Trayer I.P. Ruegg J.C. FEBS Lett. 1995; 377: 131-134Crossref PubMed Scopus (40) Google Scholar) that the are not in unphosphorylated troponin. Our suggests that residues in both regions and 24–29 to binding to We and for help with the experiments and and for We also Trayer and for and the for