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• W2141011888 abstract "The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties. Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca2+-binding properties compared with skeletal TnC and features a Ca2+-binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca2+-saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a “closed” conformation even in the Ca2+-bound (the “on”) state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC. This structure in the Ca2+-bound state, and its subsequent interaction with troponin I (TnI), are crucial in determining the specific regulatory mechanism for cardiac muscle contraction. Further, it will allow for an understanding of the action of calcium-sensitizing drugs, which bind to cardiac TnC and are known to enhance the ability of cardiac TnC to activate cardiac muscle contraction. The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties. Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca2+-binding properties compared with skeletal TnC and features a Ca2+-binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca2+-saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a “closed” conformation even in the Ca2+-bound (the “on”) state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC. This structure in the Ca2+-bound state, and its subsequent interaction with troponin I (TnI), are crucial in determining the specific regulatory mechanism for cardiac muscle contraction. Further, it will allow for an understanding of the action of calcium-sensitizing drugs, which bind to cardiac TnC and are known to enhance the ability of cardiac TnC to activate cardiac muscle contraction. Transient increases in cytosolic Ca2+ levels in the cardiac muscle cell must be recognized by the thin filament to regulate cardiac muscle contraction. This critical function is accomplished by cardiac TnC 1The abbreviations used are: TnC, troponin C; TnI, troponin I; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; MOPS, 4-morpholinepropanesulfonic acid. 1The abbreviations used are: TnC, troponin C; TnI, troponin I; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; MOPS, 4-morpholinepropanesulfonic acid. (161 residues), a member of the EF-hand family of Ca2+-binding proteins, which relays the Ca2+ signal via a conformational change to the rest of the troponin-tropomyosin complex, and ultimately signals the activation of the myosin-actin ATPase reaction. Although the sequence of cardiac TnC is 70% identical to that of skeletal TnC, there are significant differences in the first 40 residues, the most crucial being the inactivation of Ca2+-binding site I due to an insertion (Val28) and substitutions of key ligands relative to skeletal TnC (Leu29 and Ala31 in cardiac TnC instead of Asp30 and Asp32 in skeletal TnC) (1Van Eerd J.P. Takahashi K. Biochem. Biophys. Res. Commun. 1975; 64: 122-127Crossref PubMed Scopus (116) Google Scholar). Despite the many functional, binding, and modeling studies performed on cardiac TnC (2Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (454) Google Scholar), the absence of direct structural data makes the Ca2+-induced conformational change in cardiac TnC unclear. The structures of TnC in the skeletal system, on the other hand, have been solved both in the 2-Ca2+ (3Herzberg O. James M.N.G. Nature. 1985; 313: 653-659Crossref PubMed Scopus (482) Google Scholar, 4Sundaralingam M. Bergstrom R. Strasburg G. Rao S.T. Roychowdhury P. Greaser M. Wang B.C. Science. 1985; 227: 945-948Crossref PubMed Scopus (282) Google Scholar) and 4-Ca2+ states (5Slupsky C.M. Sykes B.D. Biochemistry. 1995; 34: 15953-15964Crossref PubMed Scopus (185) Google Scholar), showing TnC to be a dumbbell-shaped molecule with separate N- and C-terminal domains connected by a central linker. Upon Ca2+ binding, the regulatory N-domain of skeletal TnC switches from a “closed” to an “open” conformation, thereby exposing a patch of hydrophobic residues, which is thought to interact with skeletal TnI (6Gagné S.M. Tsuda S. Li M.X. Smillie L.B. Sykes B.D. Nat. Struct. Biol. 1995; 2: 784-789Crossref PubMed Scopus (250) Google Scholar). In this report, we show that, in contrast to predicted models (7Rao V.G. Akella A.B. Su H. Gulati J. Biochemistry. 1995; 34: 562-568Crossref PubMed Scopus (7) Google Scholar, 8Pollesello P. Ovaska M. Kaivola J. Tilgmann C. Lundstrom K. Kalkkinen N. Ulmanen I. Nissinen E. Taskinen J. J. Biol. Chem. 1994; 269: 28584-28590Abstract Full Text PDF PubMed Google Scholar, 9Brito R.M.M. Putkey J.A. Strynadka N.C.J. James M.N.G. Rosevear P.R. Biochemistry. 1991; 30: 10236-10245Crossref PubMed Scopus (26) Google Scholar), the analogous conformational change does not occur in cardiac TnC, and that this is the direct structural consequence of inactivating Ca2+-binding site I. In addition, a structural understanding of cardiac TnC has potential therapeutic value in the understanding of the mechanism of cardiac TnC-binding drugs known as “calcium-sensitizing drugs” (8Pollesello P. Ovaska M. Kaivola J. Tilgmann C. Lundstrom K. Kalkkinen N. Ulmanen I. Nissinen E. Taskinen J. J. Biol. Chem. 1994; 269: 28584-28590Abstract Full Text PDF PubMed Google Scholar, 10Endoh M. Gen. Pharmacol. 1995; 26: 1-31Crossref PubMed Scopus (80) Google Scholar).For the purposes of this study, the two Cys residues at positions 35 and 84 of wild type cardiac TnC have been mutated to Ser residues. This prevents the formation of intra- and intermolecular disulfide bonds, which confer Ca2+-independent activity to cardiac TnC when assayed in skeletal muscle myofibrils (11Putkey J.A. Dotson D.G. Mouawad P. J. Biol. Chem. 1993; 268: 6827-6830Abstract Full Text PDF PubMed Google Scholar). It has been shown that the conversion of these Cys residues to Ser residues has no effect on the ability of cardiac TnC to recover ATPase activity in TnC-extracted fast skeletal and cardiac myofibrils, and has little effect on Ca2+ binding to site II of cardiac TnC (11Putkey J.A. Dotson D.G. Mouawad P. J. Biol. Chem. 1993; 268: 6827-6830Abstract Full Text PDF PubMed Google Scholar). Thus, it is unlikely that the introduction of these two conservative mutations would result in gross conformational changes in the secondary or tertiary structure of cardiac TnC.For NMR analysis, the protein was uniformly labeled with13C and/or 15N by expression inEscherichia coli. Triple-resonance NMR experiments were used for assigning the resonances and subsequently to derive distance and dihedral angle restraints. 35 structures were then calculated using the simulated annealing protocol (12Nilges M. Gronenborn A.M. Brunger A.T. Clore G.M. Protein Eng. 1988; 2: 27-38Crossref PubMed Scopus (513) Google Scholar). Structural statistics for the 30 lowest energy structures (Table I) show that the N- and C-domains are very well defined separately, with the central linker shown to be flexible by relaxation measurements. 2S. K. Sia, M. X. Li, L. Spyracopoulos, S. M. Gagné, W. Liu, J. A. Putkey, and B. D. Sykes, unpublished data. 2S. K. Sia, M. X. Li, L. Spyracopoulos, S. M. Gagné, W. Liu, J. A. Putkey, and B. D. Sykes, unpublished data.Table IStructural statisticsN-domain (2–89)C-domain (90–161)r.m.s.d. from the average structure (Å)1-a30 structures were calculated with the method of simulated annealing (12), using the program X-PLOR (35). Because the central linker (85–94) is unstructured, the structures of the N- and C-domains were calculated separately. Root-mean-squared deviations (r.m.s.d.) are for residues 5–29, 34–48, 56–65, and 69–84 for the N-domain, and residues 97–124 and 130–158 for the C-domain. Backbone atoms0.54 ± 0.090.46 ± 0.07 All heavy atoms0.97 ± 0.090.94 ± 0.10NOE restraints Total12391080 Intra-residue 477 421 Sequential (‖i− j| = 1) 285 267 Medium-range (2 ≤ ‖i − j| ≤ 4) 315 222 Long-range (‖i − j| ≥ 5) 162 170Distance restraints to Ca2+-ion1-bNote that calcium ions are not directly observed by NMR spectroscopy. No restraints involving the calcium ions were used in the initial stages of structure calculations, and were added only in the final stages of refinement (see “Materials and Methods”).612Dihedral restraints Total 10487 φ4941 ψ3726 χ11820Energies1-cThe final force constants were K NOE = 50 kcal mol−1 and K dihedral = 200 kcal mol−1rad−2. φ, ψ core and allowed regions were as determined by the program PROCHECK (36). E total105 ± 484 ± 2 E NOE2 ± 14 ± 1 E dihedral0.03 ± 0.030.03 ± 0.05r.m.s.d. from idealized geometry bond lengths (Å)0.0012 ± 0.00010.0011 ± 0.0001 bond angle (°)0.47 ± 0.010.44 ± 0.01 impropers (°)0.35 ± 0.010.33 ± 0.01Restraint violations distance > 0.1 Å but < 0.2 Å1-dThere are no distance violations over 0.2 Å for the N-domain, and there is 1 distance violation over 0.2 Å for 30 structures for the C-domain.52 (1.7/structure)50 (1.7/structure) dihedral > 1° 0 (0/structure) 0 (0/structure)φ, ψ in core or allowed regions98%99%1-a 30 structures were calculated with the method of simulated annealing (12Nilges M. Gronenborn A.M. Brunger A.T. Clore G.M. Protein Eng. 1988; 2: 27-38Crossref PubMed Scopus (513) Google Scholar), using the program X-PLOR (35Brunger A.T. X-PLOR 3.1 Manual. Yale University Press, New Haven, CT1992Google Scholar). Because the central linker (85–94) is unstructured, the structures of the N- and C-domains were calculated separately. Root-mean-squared deviations (r.m.s.d.) are for residues 5–29, 34–48, 56–65, and 69–84 for the N-domain, and residues 97–124 and 130–158 for the C-domain.1-b Note that calcium ions are not directly observed by NMR spectroscopy. No restraints involving the calcium ions were used in the initial stages of structure calculations, and were added only in the final stages of refinement (see “Materials and Methods”).1-c The final force constants were K NOE = 50 kcal mol−1 and K dihedral = 200 kcal mol−1rad−2. φ, ψ core and allowed regions were as determined by the program PROCHECK (36Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-290Crossref Google Scholar).1-d There are no distance violations over 0.2 Å for the N-domain, and there is 1 distance violation over 0.2 Å for 30 structures for the C-domain. Open table in a new tab RESULTSFig. 1 shows the solution structures of the N- and C-terminal domains, with the structural statistics provided in TableI. The overall solution structure of Ca2+-saturated cardiac TnC, like the solution structures of Ca2+-saturated skeletal TnC (5Slupsky C.M. Sykes B.D. Biochemistry. 1995; 34: 15953-15964Crossref PubMed Scopus (185) Google Scholar) and calmodulin-target peptide complex (26Ikura I. Clore G.M. Gronenborn A.M. Zhu G. Klee C.B. Bax A. Science. 1992; 256: 632-638Crossref PubMed Scopus (1176) Google Scholar), resembles a dumbbell in shape, consisting of two separate domains connected by a flexible central linker (residues 86–94 in cardiac TnC). However, despite the general structural similarities to homologous Ca2+-binding proteins, the regulatory N-domain of Ca2+-saturated cardiac TnC is significantly more compact than the N-domain of Ca2+-saturated skeletal TnC (5Slupsky C.M. Sykes B.D. Biochemistry. 1995; 34: 15953-15964Crossref PubMed Scopus (185) Google Scholar, 6Gagné S.M. Tsuda S. Li M.X. Smillie L.B. Sykes B.D. Nat. Struct. Biol. 1995; 2: 784-789Crossref PubMed Scopus (250) Google Scholar), exposing approximately 800 Å2 less total accessible surface area (residues 5–84) than its skeletal counterpart (residues 7–85). In particular, the B-helix of defunct site I exists in the “closed” conformation, exhibiting an A-B interhelical angle of 142° (with the A- and B-helices corresponding to the two helices of the helix-loop-helix motif in Ca2+-binding proteins; Table II). The closed conformation is evidenced by 21 NOE connectives observed between the A- and B-helices (Fig. 2), most of which would not be observed if the B-helix were in an “open” conformation as in skeletal TnC (Fig. 3 A). A compact regulatory domain is also consistent with a previous cysteine-reactivity study on wild type cardiac TnC (27Fuchs F. Liou Y.-M. Grabarek Z. J. Biol. Chem. 1989; 264: 20344-20349Abstract Full Text PDF PubMed Google Scholar).Table IIInterhelical angles of various EF-handsCalcium-binding protein2-aThe parentheses indicate first the state of the N-domain (i.e., A-B and C-D helix-loop-helices), followed by the state of the C-domain (i.e., E-F and G-H helix-loop-helices). Note that in cardiac TnC(1 Ca2+/2 Ca2+) as determined in the present study, defunct site I (i.e. A-B helix-loop-helix) is free of Ca2+, while sites II, III, and IV are Ca2+-bound. Protein Data Bank accession codes are: 1TNW for the NMR structure of skeletal TnC(2 Ca2+/2 Ca2+), 5TNC for the crystal structure of skeletal TnC(apo/2 Ca2+), and 4CLN for the crystal structure of calmodulin(2 Ca2+/2 Ca2+).Interhelical angles (°)2-bA large angle defines a “closed” conformation, whereas a small angle defines an “open” conformation. The axis for an α-helix is defined by two points, the two points being the average coordinates of the first and last 11 backbone atoms of the α-helix.A-BC-DE-FG-HCardiac TnC(1 Ca2+/2 Ca2+)138 ± 3108 ± 4115 ± 4121 ± 4Skeletal TnC(2 Ca2+/2 Ca2+)81 ± 578 ± 789 ± 6104 ± 7Skeletal TnC(apo/2 Ca2+)138145105111Calmodulin(2 Ca2+/2 Ca2+) 88 92 98 972-a The parentheses indicate first the state of the N-domain (i.e., A-B and C-D helix-loop-helices), followed by the state of the C-domain (i.e., E-F and G-H helix-loop-helices). Note that in cardiac TnC(1 Ca2+/2 Ca2+) as determined in the present study, defunct site I (i.e. A-B helix-loop-helix) is free of Ca2+, while sites II, III, and IV are Ca2+-bound. Protein Data Bank accession codes are: 1TNW for the NMR structure of skeletal TnC(2 Ca2+/2 Ca2+), 5TNC for the crystal structure of skeletal TnC(apo/2 Ca2+), and 4CLN for the crystal structure of calmodulin(2 Ca2+/2 Ca2+).2-b A large angle defines a “closed” conformation, whereas a small angle defines an “open” conformation. The axis for an α-helix is defined by two points, the two points being the average coordinates of the first and last 11 backbone atoms of the α-helix. Open table in a new tab Figure 2Evidence for the closed conformation for the regulatory domain. A, three-dimensional15N/13C-edited NOESY showing 4 of the 21 NOE contacts between the A- and B-helices. Here, the Hβ protons of Ala23 show NOE connectives to the Hδ1 and Hδ2 protons of Leu48 and to the Hγ1 and Hγ2 protons of Val44. Negative (folded) peaks are indicated bydotted lines. B, structure of the A-helix-loop-B-helix (residues 14–49) of the regulatory domain. Thefour dotted lines correspond to the four NOEs shown inA. This figure was prepared with the software INSIGHT II (Biosym).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3A, comparison of the regulatory N-domains of TnC in various states. Shown here are 2Ca·skeletal TnC (green, left panel; neither site occupied by Ca2+), 3Ca·cardiac TnC (red, middle panel; site II occupied by Ca2+ and site I unable to bind Ca2+), and 4Ca·skeletal TnC (blue,right panel; both sites I and II occupied by Ca2+). The B- and C-helices of all three structures are shown in gray. The residue E40 is labeled by anarrow. B, comparison of various structural C-domains of TnC, all in the Ca2+-saturated state (both sites III and IV occupied by Ca2+). Shown here are 2Ca·skeletal TnC (green, left panel), 3Ca·cardiac TnC (red, middle panel), and 4Ca·skeletal TnC (blue, right panel). This figure was prepared with the program RASTER3D (37Merritt E.A. Murphy M.E.P. Acta. Crystallogr. D. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The difference in the conformation of the B-helix is reflected most clearly in the main chain conformation of residue Glu40 in cardiac TnC (equivalent to Glu41 in skeletal TnC). Glu41 of skeletal TnC has been proposed to be pivotal in the mechanism of the coupling between Ca2+ binding and the Ca2+-induced conformational change in Ca2+-binding proteins (28Gagné 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, 29Gagné S.M. Li M.X. Sykes B.D. Biochemistry. 1997; 36: 4386-4392Crossref PubMed Scopus (113) Google Scholar). Indeed, the apo form of skeletal TnC (3Herzberg O. James M.N.G. Nature. 1985; 313: 653-659Crossref PubMed Scopus (482) Google Scholar) features a kink in the B-helix at Glu41which straightens out upon Ca2+ binding to sites I and II (28Gagné 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). In cardiac TnC, however, there exists a kink in the B-helix at Glu40, even in the Ca2+-bound state (Ca2+ ions bound at sites II, III, and IV). The non-helical nature at Glu40 is supported by a3JHNHα value of 7.8 Hz, an absence of an upfield-shift of its Hα resonance (4.37 ppm), and an absence of a downfield shift in its Cα resonance (56.8 ppm), all of which indicate non-helical conformations (30Wishart D.S. Sykes B.D. Richards F.M. J. Mol. Biol. 1991; 222: 311-333Crossref PubMed Scopus (1781) Google Scholar). On the other hand, both adjacent residues Lys39 and Leu41 exhibit3JHNHα values of less than 5.5 Hz, as well as appropriate shifts in their Hα and Cα resonances which indicate an α-helical conformation.The above structural differences between cardiac and skeletal TnC can be explained by what is in fact the most striking functional difference between the two proteins: namely that site I in cardiac TnC is inactive due to an insertion and key substitutions of key ligands. In cardiac TnC, there is no Ca2+ at site I to pull the Glu40 side-chain carboxylate group over to contribute to the enthalpy necessary to overcome the entropic loss associated with exposing buried residues (which occurs in the “opening” of the B- and C-helices relative to helices N, A, and D). Thus, the B-helix remains closed due to favorable packing forces with the A-helix and D-helix (Figs. 1 A and 3 A; Table II, A-B interhelical angle). On the other hand, with Ca2+ bound at site II, the C-helix is in fact in an open conformation, but does not open up to the extent seen in skeletal TnC (Figs. 1 A and3 A; Table II, C-D interhelical angle). These observations demonstrate that the inability of Glu40 to coordinate Ca2+ results in a more compact conformation for the B-helix, and possibly the C-helix, than is observed in skeletal TnC (Fig. 3 A); in effect, Ca2+ binding to sites I and II of skeletal TnC locks open the whole regulatory domain, whereas Ca2+ binding to site II of cardiac TnC only partially opens up the regulatory domain. (This discussion assumes that the structure of the apo form of the regulatory domain of cardiac TnC is similar to that of skeletal TnC, as has been recently demonstrated. 3L. Spyracopoulos, M. X. Li, S. K. Sia, S. M. Gagné, M. Chandra, R. J. Solaro, and B. D. Sykes, unpublished data. ) The model of Glu40 acting as a pivot for the N-domain is further supported by a recent structural study of a skeletal TnC mutant in which Glu41 is replaced by Ala41, such that residue 41 can no longer coordinate the Ca2+ ion present at site I (29Gagné S.M. Li M.X. Sykes B.D. Biochemistry. 1997; 36: 4386-4392Crossref PubMed Scopus (113) Google Scholar). In the Ca2+-saturated state of this protein, the single substitution results in a kink at Ala41 and a closed conformation for the B-helix, similar to what is seen in Ca2+-saturated cardiac TnC.The structure of defunct site I shows that Leu29, which comes just after the insertion at Val28, forms an extra half-turn at the end of the A-helix, as evidenced by dαN(i, i + 3) and dαβ(i, i + 3) NOE connectives from Ile26 to Leu29. Site I, being Ca2+-free, is not as well defined as the rest of the molecule (root mean square deviation of 0.78 Å for backbone atoms of residues 30–33), and is shown to be more flexible than the rest of the regulatory domain by relaxation measurements.The structural C-domain of cardiac TnC (Figs. 1 B and3 B) is predictably similar to those in skeletal TnC and calmodulin, although the interhelical angles of the two EF-hands in the C-domain indicate that this domain is in fact slightly more compact in cardiac TnC than in its counterparts (10–20° more closed in the E-F and G-H interhelical angles; see Table II). Overall, the backbone atoms of residues 95–157 of cardiac TnC superimpose within 1.9 Å with their equivalent residues (96–158) in the NMR structure of skeletal TnC with Ca2+-saturated N-domain, and 1.3 Å with the same region in the crystal structure of skeletal TnC with apo N-domain.DISCUSSIONWe have shown for the first time the three-dimensional structure of Ca2+-saturated cardiac TnC, which reveals an unexpected compact regulatory domain as a direct consequence of an inactive Ca2+-binding site I. These results provide a structural precedent for a Ca2+-binding regulatory protein in which one of the two sites in the paired set of EF-hands is inactive (for example, some invertebrate TnCs also have this feature; Ref. 31Kobayashi T. Takagi T. Konishi K. Wnuk W. J. Biol. Chem. 1989; 264: 18247-18259Abstract Full Text PDF PubMed Google Scholar)). This unique structural feature sets cardiac TnC apart from other “calcium sensor” EF-hand Ca2+-binding proteins such as skeletal TnC and calmodulin, as well as “calcium buffer” EF-hand proteins such as parvalbumin and calbindin. The compact regulatory domain is a surprising result because it violates the general rule with Ca2+-binding proteins that a small conformational change accompanies Ca2+ binding in buffering proteins, and that a large conformational change accompanies Ca2+ binding in regulatory proteins such as cardiac TnC (32Skelton J.N. Kördel J. Akke M. Forsén S. Chazin W.J. Nat. Struct. Biol. 1994; 1: 239-245Crossref PubMed Scopus (147) Google Scholar). In particular, it is believed that in general the action of Ca2+ binding in calcium sensor proteins is to induce an exposure of a large hydrophobic surface, allowing the protein to interact with targets to accomplish regulatory functions, whereas the capture of Ca2+ ions by calcium buffer proteins is accompanied by only minor conformational changes. Thus, it has long been believed that the mechanism for the activation of cardiac TnC involves the exposure of a large hydrophobic patch upon Ca2+ binding as observed for other calcium sensors. In fact, cardiac TnC models based on the conformational changes observed in skeletal TnC have been widely used to interpret the functional, Ca2+-binding and drug-binding properties of cardiac TnC (7Rao V.G. Akella A.B. Su H. Gulati J. Biochemistry. 1995; 34: 562-568Crossref PubMed Scopus (7) Google Scholar, 8Pollesello P. Ovaska M. Kaivola J. Tilgmann C. Lundstrom K. Kalkkinen N. Ulmanen I. Nissinen E. Taskinen J. J. Biol. Chem. 1994; 269: 28584-28590Abstract Full Text PDF PubMed Google Scholar, 9Brito R.M.M. Putkey J.A. Strynadka N.C.J. James M.N.G. Rosevear P.R. Biochemistry. 1991; 30: 10236-10245Crossref PubMed Scopus (26) Google Scholar), despite the unique inactive Ca2+-binding site I in cardiac TnC. The present results show that the hydrophobic exposure in the Ca2+-saturated regulatory domain of cardiac TnC (Fig.4 B) is dramatically reduced compared with that of skeletal TnC (Fig. 4 A) as well as a previous widely used model of cardiac TnC (9Brito R.M.M. Putkey J.A. Strynadka N.C.J. James M.N.G. Rosevear P.R. Biochemistry. 1991; 30: 10236-10245Crossref PubMed Scopus (26) Google Scholar) (Fig. 4 C).Figure 4Comparison of the surface structures of the regulatory N-domains of 4Ca·skeletal TnC (NMR) (A), 3Ca·cardiac TnC (NMR) (B), and 3Ca·cardiac TnC (model, Ref. 9Brito R.M.M. Putkey J.A. Strynadka N.C.J. James M.N.G. Rosevear P.R. Biochemistry. 1991; 30: 10236-10245Crossref PubMed Scopus (26) Google Scholar) (C), displayed in the same orientation as Fig.1 A. Side chains of hydrophobic residues (Ala, Ile, Leu, Met, Pro, Phe, Tyr, and Val) are shown in yellow, negatively charged residues (Asp and Glu) in red, positively charged residues (Arg and Lys) in blue, and all other residues in gray. The major hydrophobic pocket of 3Ca·cardiac TnC involves residues Phe20, Phe24, Leu48, Phe74, Phe77, Leu78, Met81, and Met85, and residues Ile36, Leu41, Met45, Leu57, Met60, Ile61, Val64, Val72, and Met80. Other hydrophobic contacts are also observed from Phe20, Ala23, and Phe27 of the A-helix to Val44, Met47, and Leu48of the B-helix, and from Ala8, Val9, and Leu12 of the N-helix to Leu78, Val79, and Val82 of the D-helix. This figure was generated using the program GRASP (38Nicholls A. GRASP. Columbia University, New York, NY1992Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The substantially reduced hydrophobic surface of Ca2+-saturated cardiac TnC has important implications for the association of cardiac TnI with cardiac TnC. In particular, given that in both the cardiac and skeletal systems the Ca2+-dependent binding of TnI involves the N-domain of TnC (33Krudy G.A. Kleerekoper Q. Guo X. Howarth J.W. Solaro R.J. Rosevear P.R. J. Biol. Chem. 1994; 269: 23731-23735Abstract Full Text PDF PubMed Google Scholar), and that residues 5–84 of Ca2+-saturated cardiac TnC expose less total and hydrophobic surface area than residues 7–85 of Ca2+-saturated skeletal TnC (Fig. 4), it is possible that the mode of interaction between TnI and TnC in cardiac muscle is in fact different from that in skeletal muscle. A smaller surface of interaction between 3Ca·cardiac TnC-cardiac TnI as compared with 4Ca·skeletal TnC-skeletal TnI would explain the finding that the free energy (ΔG) of Ca2+ binding to the TnC-TnI complex is 4 times smaller in cardiac than it is in skeletal muscle (34Liao R. Wang C.K. Cheung H.C. Biochemistry. 1994; 33: 12729-12734Crossref PubMed Scopus (58) Google Scholar). It may also be that the hydrophobic or electrostatic force dominates more in one isoform than in the other in the binding of TnI to TnC. If indeed the interaction between cardiac TnC and cardiac TnI involves less surface contact than that between skeletal TnC and skeletal TnI, cardiac TnC would be a more dynamic calcium sensor than its skeletal counterpart. In fact, a recent study has shown that the Ca2+ off rate measured for site II in cardiac TnC is about 3-fold faster than observed for the N-terminal signaling domain of calmodulin or skeletal troponin C. 4A. L. Hazard, N. L. Stricker, J. A. Putkey, and J. J. Falke, unpublished data. On the other hand, as an alternative to the above proposal, it is possible that the Ca2+-dependent binding of TnI forces open the regulatory domain of cardiac TnC, with the end result being that the cardiac TnI-TnC complex binds in a similar fashion to skeletal TnI-TnC (here cardiac TnC may adopt a structure similar to that in Fig.4 C). Such a model would be consistent with the finding that the chemical environment of Met81, which is mostly buried in this structure (accessible surface area of 14 Å2), changes upon the binding of cardiac TnI (33Krudy G.A. Kleerekoper Q. Guo X. Howarth J.W. Solaro R.J. Rosevear P.R. J. Biol. Chem. 1994; 269: 23731-23735Abstract Full Text PDF PubMed Google Scholar). This may also imply that cardiac TnC opens up to different degrees in response to events of muscle contraction such as TnI phosphorylation. At present, there is no compelling evidence to either favor or discount either model for cardiac TnI-TnC binding.Cardiac TnC is a potential target in therapy for patients with acute myocardial infarctions and subsequently congestive heart failure, where the diseased myocardium is “desensitized” to increases in cytosolic Ca2+ levels. A novel group of positive inotropic agents known as “calcium sensitizers” (10Endoh M. Gen. Pharmacol. 1995; 26: 1-31Crossref PubMed Scopus (80) Google Scholar) is known to increase the affinity of cardiac TnC for Ca2+, possibly by binding to a hydrophobic patch in the N-domain of cardiac TnC (8Pollesello P. Ovaska M. Kaivola J. Tilgmann C. Lundstrom K. Kalkkinen N. Ulmanen I. Nissinen E. Taskinen J. J. Biol. Chem. 1994; 269: 28584-28590Abstract Full Text PDF PubMed Google Scholar). The exposed hydrophobic patches in Ca2+-saturated cardiac TnC can now be identified (Fig. 4 B). Although several residues (e.g. Phe77, Met81, and Met85) have been implicated in earlier studies as possible binding sites for these drugs (8Pollesello P. Ovaska M. Kaivola J. Tilgmann C. Lundstrom K. Kalkkinen N. Ulmanen I. Nissinen E. Taskinen J. J. Biol. Chem. 1994; 269: 2858" @default.
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• W2141011888 title "Structure of Cardiac Muscle Troponin C Unexpectedly Reveals a Closed Regulatory Domain" @default.
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