Cross-linking between the regulatory regions of troponin-I and troponin-C abolishes the inhibitory function of troponin

We reported previously that both residues 48 and 82 on opposite sides of troponin-C's (TnC's) N-terminal regulatory hydrophobic cleft photo-cross-linked to Met121 of troponin-I (TnI) [Luo, Y., Leszyk, J., Qian, Y., Gergely, J., and Tao, T. (1999) Biochemistry 38, 6678-6688]. Here we report that the Ca2+-absent inhibitory activity of troponin (Tn) was progressively lost as the extent of photo-cross-linking increased. To extend these studies, we constructed a mutant TnI with a single cysteine at residue 121 (TnI121). In Tn complexes containing TnI121 and mutant TnCs with a single cysteine at positions 12, 48, 82, 98, or 125 (TnC12, TnC48 etc.), TnI121 formed disulfide cross-links primarily with TnC48 and TnC82 when Ca2+ was present, and with only TnC48 when Ca2+ was absent. These results indicate that TnI Met121 is situated within the N-domain hydrophobic cleft of TnC in the presence of Ca2+, and that it moves out of the cleft upon Ca2+ removal but remains within the vicinity of TnC. Activity assays revealed that the Met121 to Cys mutation in TnI121 reduced the Ca2+-present activation of Tn, indicating that Met121 is important in hydrophobic interactions between this TnI region and TnC's N-domain cleft. The formation of a disulfide cross-link between TnI121 and TnC48 or TnC82 abolished the Ca2+-absent inhibitory activity of Tn, indicating that the movement of the Met121 region of TnI out of TnC's N-domain cleft is essential for the occurrence of further events in the inhibitory process of skeletal muscle contraction. On the basis of these and other results, a simple mechanism for Ca2+ regulation of skeletal muscle contraction is presented and discussed.     

Proximity relationships between residue 6 of troponin I and residues in troponin C: further evidence for extended conformation of troponin C in the troponin complex

Skeletal muscle troponin C (TnC) adopts an extended conformation when crystallized alone and a compact one when crystallized with an N-terminal troponin I (TnI) peptide, TnI(1-47) [Vassylyev et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4847-4852]. The N-terminal region of TnI (residues 1-40) was suggested to play a functional role of facilitating the movement of TnI's inhibitory region between TnC and actin [Tripet et al. (1997) J. Mol. Biol. 271, 728-750]. To test this hypothesis and to investigate the conformation of TnC in the intact troponin complex and in solution, we attached fluorescence and photo-cross-linking probes to a mutant TnI with a single cysteine at residue 6. Distances from this residue to residues of TnC were measured by the fluorescence resonance energy transfer technique, and the sites of photo-cross-linking in TnC were determined by microsequencing and mass spectrometry following enzymatic digestions. Our results show that in the troponin complex neither the distance between TnI residue 6 and TnC residue 89 nor the photo-cross-linking site in TnC, Ser133, changes with Ca(2+), in support of the notion that this region plays mainly a structural rather than a regulatory role. The distances to residues 12 and 41 in TnC's N-domain are both considerably longer than those predicted by the crystal structure of TnC.TnI(1-47), supporting an extended rather than a compact conformation of TnC. In the binary TnC.TnI complex and the presence of Ca(2+), Met43 in TnC's N-domain was identified as the photo-cross-linking site, and multiple distances between TnI residue 6 and TnC residue 41 were detected. This was taken to indicate increased flexibility in TnC's central helix and that TnC dynamically changes between a compact and an extended conformation when troponin T (TnT) is absent. Our results further emphasize the difference between the binary TnC.TnI and the ternary troponin complexes and the importance of using intact proteins in the study of structure-function relationships of troponin.     

Proximity relationships between residue 117 of rabbit skeletal troponin-I and residues in troponin-C and actin.

We used resonance energy transfer and site-directed photo-cross-linking to probe the Ca(2+)-dependent proximity relationships between residue 117 next to the C-terminus of the inhibitory region in rabbit skeletal troponin-I (TnI) and residues in troponin-C (TnC) and in actin. A mutant TnI that contains a single cysteine at position 117 (I117) was constructed, and the distance between TnI residue 117 and TnC residue 98 was measured with the following results: for both the binary TnC-TnI complex and the ternary troponin complex, this distance was 30 and 41 A in the presence and absence of Ca(2+), respectively. The distance between TnI residue 117 and Cys374 of actin was 48 and 41 A in the presence and absence of Ca(2+), respectively. Six additional distances from this TnI residue to cysteines in TnC mutants were measured and used to localize this residue with respect to the crystal structure of TnC. The results show that in the presence of Ca(2+) it is localized near the B and C helices of TnC's N-terminal domain. In the absence of Ca(2+) this residue moves away from this location by approximately 8 A. Photo-cross-linking experiments show that I117 labeled with 4-maleimidobenzophenone photo-cross-linked to TnC but not to actin in both the presence and absence of Ca(2+). Taken together these results provide independent experimental support for the proposal (Y. Luo, J. L. Wu, B. Li, K. Langsetmo, J. Gergely, and T. Tao, 2000, J. Mol. Biol. 296:899-910) that upon Ca(2+) removal the region comprising TnI residues 114-125 triggers the movements of residues 89-113 and 130-150 toward actin, but does not itself interact with actin.     

Photocrosslinking of benzophenone-labeled single cysteine troponin I mutants to other thin filament proteins

The interaction sites of rabbit skeletal troponin I (TnI) with troponin C (TnC), troponin T (TnT), tropomyosin (Tm) and actin were mapped systematically using nine single cysteine residue TnI mutants with mutation sites at positions 6, 48, 64, 89, 104, 121, 133, 155 or 179 (TnI6, TnI48 etc.). Each mutant was labeled with the heterobifunctional photocrosslinker 4-maleimidobenzophenone (BP-Mal), and incorporated into the TnI.TnC binary complex, the TnI.TnC.TnT ternary troponin (Tn) complex, and the Tn.Tm.F-actin synthetic thin filament. Photocrosslinking reactions carried out in the presence and absence of Ca(2+) yielded the following results: (1) BP-TnI6 photocrosslinked primarily to TnC with a small degree of Ca(2+)-dependence in all the complex forms. (2) BP-TnI48, TnI64 and TnI89 photocrosslinked to TnT with no Ca(2+)-dependence. Photocrosslinking to TnC was reduced in the ternary versus the binary complex. BP-TnI89 also photocrosslinked to actin with higher yields in the absence of Ca(2+) than in its presence. (3) BP-TnI104 and TnI133 photocrosslinked to actin with much higher yields in the absence than in the presence of Ca(2+). (4) BP-TnI121 photocrosslinked to TnC with a small degree of Ca(2+)-dependence, and did not photocrosslink to actin. (5) BP-TnI155 and TnI179 photocrosslinked to TnC, TnT and actin, but all with low yields. All the labeled mutants photocrosslinked to TnC with varying degrees of Ca(2+)-dependence, and none to Tm. These results, along with those published allowed us to construct a structural and functional model of TnI in the Tn complex: in the presence of Ca(2+), residues 1-33 of TnI interact with the C-terminal domain hydrophobic cleft of TnC, approximately 48-89 with TnT, approximately 90-113 with TnC's central helix, approximately 114-125 with TnC's N-terminal domain hydrophobic cleft, and approximately 130-150 with TnC's A-helix. In the absence of Ca(2+), residues approximately 114-125 move out of TnC's N-terminal domain hydrophobic cleft and trigger the movements of residues approximately 89-113 and approximately 130-150 away from TnC and towards actin.     

Calcium- and magnesium-dependent interactions between the C-terminus of troponin I and the N-terminal, regulatory domain of troponin C

The muscle thin filament protein troponin (Tn) regulates contraction of vertebrate striated muscle by conferring Ca2+ sensitivity to the interaction of actin and myosin. Troponin C (TnC), the Ca2+ binding subunit of Tn contains two homologous domains and four divalent cation binding sites. Two structural sites in the C-terminal domain of TnC bind either Ca2+ or Mg2+, and two regulatory sites in the N-terminal domain are specific for Ca2+. Interactions between TnC and the inhibitory Tn subunit troponin I (TnI) are of central importance to the Ca2+ regulation of muscle contraction and have been intensively studied. Much remains to be learned, however, due mainly to the lack of a three-dimensional structure for TnI. In particular, the role of amino acid residues near the C-terminus of TnI is not well understood. In this report, we prepared a mutant TnC which contains a single Trp-26 residue in the N-terminal, regulatory domain. We used fluorescence lifetime and quenching measurements to monitor Ca2+- and Mg2+-dependent changes in the environment of Trp-26 in isolated TnC, as well as in binary complexes of TnC with a Trp-free mutant of TnI or a truncated form of this mutant, TnI(1-159), which lacked the C-terminal 22 amino acid residues of TnI. We found that full-length TnI and TnI(1-159) affected Trp-26 similarly when all four binding sites of TnC were occupied by Ca2+. When the regulatory Ca2+-binding sites in the N-terminal domain of TnC were vacant and the structural sites in the C-terminal domain of were occupied by Mg2+, we found significant differences between full-length TnI and TnI(1-159) in their effect on Trp-26. Our results provide the first indica- tion that the C-terminus of TnI may play an important role in the regulation of vertebrate striated muscle through Ca2+-dependent interactions with the regula- tory domain of TnC.     

Conformational changes induced in troponin I by interaction with troponin T and actin/tropomyosin.

Troponin I (TnI) is the inhibitory component of the striated muscle Ca2+ regulatory protein troponin (Tn). The other two components of Tn are troponin C (TnC), the Ca2+-binding component, and troponin T (TnT), the tropomyosin-binding component. We have used limited chymotryptic digestion to probe the local conformation of TnI in the free state, the binary TnC*TnI complex, the ternary TnC*. TnI*TnT (Tn) complex, and in the reconstituted Tn*tropomyosin*F-actin filament. The digestion of TnI alone or in the TnC*TnI complex produced initially two major fragments via a cleavage of the peptide bond between Phe100 and Asp101 in the so-called inhibitory region. In the ternary Tn complex cleavage occurred at a new site between Leu140 and Lys141. In the absence of Ca2+ this was followed by digestion of the 1-140 fragment at Leu122 and Met116. In the reconstituted thin filament the same fragments as in the case of the ternary complex were produced, but the rate of digestion was slower in the absence than in the presence of Ca2+. These results indicate firstly that in both free TnI and TnI complexed with TnC there is an exposed and flexible site in the inhibitory region. Secondly, TnT affects the conformation of TnI in the inhibitory region and also in the region that contains the 140-141 bond. Thirdly, the 140-141 region of TnI is likely to interact with actin in the reconstituted thin filament when Ca2+ is absent. These findings are discussed in terms of the role of TnI in the mechanism of thin filament regulation, and in light of our previous results [Y. Luo, J.-L. Wu, J. Gergely, T. Tao, Biochemistry 36 (1997) 13449-13454] on the global conformation of TnI.     

Calcium-induced flexibility changes in the troponin C-troponin I complex

The contraction of vertebrate striated muscle is modulated by Ca(2+) binding to the regulatory protein troponin C (TnC). Ca(2+) binding causes conformational changes in TnC which alter its interaction with the inhibitory protein troponin I (TnI), initiating the regulatory process. We have used the frequency domain method of fluorescence resonance energy transfer (FRET) to measure distances and distance distributions between specific sites in the TnC-TnI complex in the presence and absence of Ca(2+) or Mg(2+). Using sequences based on rabbit skeletal muscle proteins, we prepared functional, binary complexes of wild-type TnC and a TnI mutant which contains no Cys residues and a single Trp residue at position 106 within the TnI inhibitory region. We used TnI Trp-106 as the FRET donor, and we introduced energy acceptor groups into TnC by labeling at Met-25 with dansyl aziridine or at Cys-98 with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our distance distribution measurements indicate that the TnC-TnI complex is relatively rigid in the absence of Ca(2+), but becomes much more flexible when Ca(2+) binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, helping to release the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distributions between TnC and TnI in their binary complex.     

Involvement of conserved, acidic residues in the N-terminal domain of troponin C in calcium-dependent regulation

We have mutated eight conserved, charged amino acid residues in the N-terminal, regulatory domain of troponin C (TnC) so we could investigate their role in troponin-linked Ca2+ regulation of muscle contraction. These residues surround a hydrophobic pocket in the N-terminal domain of TnC which, when Ca2+ binds to regulatory sites in this domain, is exposed and interacts with the inhibitory region of troponin I (TnI). We constructed three double mutants (E53A/E54A, E60A/E61A, and E85A/D86A) and two single mutants (R44A and R81A) of rabbit fast skeletal muscle troponin C (TnC) in which the charged residues were replaced with neutral alanines. All five of these mutants retained TnC's ability to bind TnI in a Ca2+-dependent manner, to neutralize TnI's inhibition of actomyosin S1 ATPase activity, and to form a ternary complex with TnI and troponin T (TnT). Ternary complexes formed with TnC(R44A) or TnC(R81A) regulated actomyosin S1 ATPase activity normally, with TnI-based inhibition in the absence of Ca2+ and TnT-based activation in the presence of Ca2+. TnC(E53A/E54A) and TnC(E85A/D86A) interacted weakly with TnT, as judged by native gel electrophoresis. Ternary complexes formed with these mutants inhibited actomyosin S1 ATPase activity in both the presence and absence of Ca2+, and did not undergo Ca2+-dependent structural changes in TnI which can be detected by limited chymotryptic digestion. TnC(E60A/E61A) interacted normally with TnT. Its ternary complex showed Ca2+-dependent structural changes in TnI, inhibited actomyosin S1 ATPase in the absence of Ca2+, but did not activate ATPase in the presence of Ca2+. This is the first demonstration that selective mutation of TnC can abolish the activating effect of troponin while its inhibitory function is retained. Our results suggest the existence of an elaborate network of protein-protein interactions formed by TnI, TnT, and the N-terminal domain of TnC, all of which are important in the Ca2+-dependent regulation of muscle contraction.     

The calcium-induced switch in the troponin complex probed by fluorescent mutants of troponin I.

The Ca2+-induced transition in the troponin complex (Tn) regulates vertebrate striated muscle contraction. Tn was reconstituted with recombinant forms of troponin I (TnI) containing a single intrinsic 5-hydroxytryptophan (5HW). Fluorescence analysis of these mutants of TnI demonstrate that the regions in TnI that respond to Ca2+ binding to the regulatory N-domain of TnC are the inhibitory region (residues 96-116) and a neighboring region that includes position 121. Our data confirms the role of TnI as a modulator of the Ca2+ affinity of TnC; we show that point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC. We also discuss the possibility that the regulatory sites in the N-terminal domain of TnC might be the high affinity Ca2+-binding sites in the troponin complex.     

Structural basis for Ca2+-regulated muscle relaxation at interaction sites of troponin with actin and tropomyosin

Troponin and tropomyosin on actin filaments constitute a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within the actin-based thin filament. Troponin consists of three subunits: an inhibitory subunit (TnI), a Ca2+-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca2+-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with 15N and/or 13C. By applying NMR spectroscopy, the solution structures of a "mobile" actin-binding domain (approximately 6.1 kDa) in the troponin ternary complex (approximately 52 kDa) were determined. The mobile domain appears to tumble independently of the core domain of troponin. Ca2+-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca2+ concentrations. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-electron microscopy (cryo-EM) density map of thin filament at low [Ca2+]. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at a low Ca2+ concentration. The resultant atomic model suggests that troponin interacted electrostatically with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at a low Ca2+ concentration. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch.     

A cross-linking study of the N-terminal extension of human cardiac troponin I

Phosphorylation of the unique N-terminal extension of cardiac troponin I (TnI) by PKA modulates Ca(2+) release from the troponin complex. The mechanism by which phosphorylation affects Ca(2+) binding, however, remains unresolved. To investigate this question, we have studied the interaction of a fragment of TnI consisting of residues 1-64 (I1-64) with troponin C (TnC) by isothermal titration microcalorimetry and cross-linking. I1-64 binds extremely tightly to the C-terminal domain of TnC and weakly to the N-terminal domain. Binding to the N-domain is weakened further by phosphorylation. Using the heterobifunctional cross-linker benzophenone-4-maleimide and four separate cysteine mutants of I1-64 (S5C, E10C, I18C, R26C), we have probed the protein-protein interactions of the N-terminal extension. All four I1-64 mutants cross-link to the N-terminal domain of TnC. The cross-linking is enhanced by Ca(2+) and reduced by phosphorylation. By introducing the same monocysteine mutations into full-length TnI, we were able to probe the environment of the N-terminal extension in intact troponin. We find that the full length of the extension lies in close proximity to both TnC and troponin T (TnT). Ca(2+) enhances the cross-linking to TnC. Cross-linking to both TnC and TnT is reduced by prior phosphorylation of the TnI. In binary complexes the mutant TnIs cross-link to both the isolated TnC N-domain and whole TnC. Cyanogen bromide digestion of the covalent TnI-TnC complex formed from intact troponin demonstrates that cross-linking is predominantly to the N-terminal domain of TnC.     

Localization of Cys133 of rabbit skeletal troponin-I with respect to troponin-C by resonance energy transfer.

We have used the technique of resonance energy transfer in conjunction with distance geometry analysis to localize Cys133 of troponin-I (TnI) with respect to troponin-C (TnC) in the ternary troponin complex and the binary TnC.TnI complex in the presence and absence of Ca2+. Cys133 of TnI was chosen because our previous work has shown that the region of TnI containing this residue undergoes Ca2+-dependent movements between actin and TnC, and may play an important role in the regulatory function of troponin. For this purpose, a TnI mutant with a single Cys at position 133, and TnC mutants, each with a single Cys at positions 5, 12, 21, 41, 49, 89, 98, 133, and 158, were constructed by site-directed mutagenesis. The distances between TnI Cys133 and each of the nine residues in TnC were then measured. Using a least-squares minimization procedure, we determined the position of TnI Cys133 in the coordinate system of the crystal structure of TnC. Our results show that in the presence of Ca2+, TnI Cys133 is located near residue 12 beneath the N-terminal lobe of TnC, and moves away by 12.6 A upon the removal of Ca2+. TnI Cys133 and the region of TnC that undergoes major change in conformation in response to Ca2+ are located roughly on opposite sides of TnC's central helix. This suggests that the region in TnI that undergoes Ca2+-dependent interaction with TnC is distinct from that interacting with actin.     

Troponin I enhances acidic pH-induced depression of Ca2+ binding to the regulatory sites in skeletal troponin C

Inhibition of muscle force development by acidic pH is a well known phenomenon, yet the exact mechanism by which a decrease in pH inhibits the Ca2+-activated force in striated myofilaments remains poorly understood. Whether or not the deactivation by acidic pH involves direct competition between Ca2+ and protons for regulatory binding sites on fast skeletal troponin C (TnC) or whether other proteins in thin filament regulation are important remains unclear. We measured the effects of acidic pH on Ca2+-dependent fluorescent changes in TnC labeled with the probe danzylaziridine (Danz), which reports Ca2+ binding to the regulatory (Ca2+-specific) sites. Measurements were also made with TnCDanz complexed with the inhibitory Tn unit, TnI, and in the whole Tn complex. Our results show that a drop in pH from 7.0 to 6.5 is associated with a 1.6-fold increase in the midpoint for the relation between free Ca2+ and Ca2+ binding to the regulatory sites on TnCDanz. However, when TnCDanz was present in its complex with either TnI alone or with TnI-TnT, the increase in midpoint free Ca2+ was increased by 3.5-fold. We tested whether this potentiation in the effect of acidic pH on Ca2+ binding to TnC is due to a pH-induced alteration in the binding of TnI to TnC. A decrease in pH from 7.0 to 6.5 was associated with a halving of the affinity of TnI for TnC. We also probed the effect of acidic pH on TnI. This was done (i) by measuring the intrinsic fluorescence of tryptophan residues in TnI alone and (ii) by measuring fluorescence of TnI (in the Tn complex) labeled at Cys-133 with 5-iodoacetamidofluorescein. A drop in pH from 7.0 to 6.5 was associated with a 15% decrease in intrinsic fluorescence and with a 30% decrease in the fluorescence of the 5-iodoacetamidofluorescein probe. We conclude, therefore, that while protons and Ca2+ may directly affect Ca2+ binding to regulatory sites on fast skeletal TnC, the effect of acidic pH on TnC Ca2+ binding is amplified in the TnI-TnC and Tn complexes by a pH-related effect on the affinity of TnI for TnC.     

Effect of troponin I phosphorylation by protein kinase A on length-dependence of tension activation in skinned cardiac muscle fibers

We examined the effect of troponin I (TnI) phosphorylation by cAMP-dependent protein kinase (PKA) on the length-dependent tension activation in skinned rat cardiac trabeculae. Increasing sarcomere length shifted the pCa (-log[Ca2+])-tension relation to the left. Treatment with PKA decreased the Ca2+ sensitivity of the myofilament and also decreased the length-dependent shift of the pCa-tension relation. Replacement of endogenous TnI with phosphorylated TnI directly demonstrated that TnI phosphorylation is responsible for the decreased length-dependence. When MgATP concentration was lowered in the absence of Ca2+, tension was elicited through rigorous cross-bridge-induced thin filament activation. Increasing sarcomere length shifted the pMgATP (-log[MgATP])-tension relation to the right, and either TnI phosphorylation or partial extraction of troponin C (TnC) abolished this length-dependent shift. We conclude that TnI phosphorylation by PKA attenuates the length-dependence of tension activation in cardiac muscle by decreasing the cross-bridge-dependent thin filament activation through a reduction of the interaction between TnI and TnC.     

Site-directed spin labeling electron paramagnetic resonance study of the calcium-induced structural transition in the N-domain of human cardiac troponin C complexed with troponin I

Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggers skeletal and cardiac muscle contraction. The salient feature of this structural transition is the movement of the B and C helices, which is termed the "opening" of the N-domain. This movement exposes a hydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seen in the crystal structure of the troponin ternary complex [Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2003) Nature 424, 35-41]. In contrast to skeletal TnC, Ca(2+)-binding site I (an EF-hand motif that consists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising from comparisons with skeletal TnC is how both helices move according to Ca(2+) binding or interact with TnI in cardiac TnC. In this study, we examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directed spin labeling electron paramagnetic resonance (EPR). Doubly spin-labeled TnC mutants were prepared, and the spin-spin distances were estimated by analyzing dipolar interactions with the Fourier deconvolution method. An interspin distance of 18.4 A was estimated for mutants spin labeled at G42C on the B helix and C84 on the D helix in a Mg(2+)-saturated monomer state. The interspin distance between Q58C on the C helix and C84 on the D helix was estimated to be 18.3 A under the same conditions. Distance changes were observed by the addition of Ca(2+) ions and the formation of a complex with TnI. Our data indicated that the C helix moved away from the D helix in a distinct Ca(2+)-dependent manner, while the B helix did not. A movement of the B helix by interaction with TnI was observed. Both Ca(2+) and TnI were also shown to be essential for the full opening of the N-domain in cardiac TnC.     

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.     

Solution structure of the chicken skeletal muscle troponin complex via small-angle neutron and X-ray scattering

Troponin is a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle by participating in a series of conformational events within the actin-based thin filament. Troponin is a heterotrimeric complex consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT). Ternary troponin complexes have been produced by assembling recombinant chicken skeletal muscle TnC, TnI and the C-terminal portion of TnT known as TnT2. A full set of small-angle neutron scattering data has been collected from TnC-TnI-TnT2 ternary complexes, in which all possible combinations of the subunits have been deuterated, in both the +Ca2+ and -Ca2+ states. Small-angle X-ray scattering data were also collected from the same troponin TnC-TnI-TnT2 complex. Guinier analysis shows that the complex is monomeric in solution and that there is a large change in the radius of gyration of TnI when it goes from the +Ca2+ to the -Ca2+ state. Starting with a model based on the human cardiac troponin crystal structure, a rigid-body Monte Carlo optimization procedure was used to yield models of chicken skeletal muscle troponin, in solution, in the presence and in the absence of regulatory calcium. The optimization was carried out simultaneously against all of the scattering data sets. The optimized models show significant differences when compared to the cardiac troponin crystal structure in the +Ca2+ state and provide a structural model for the switch between +Ca2+ and -Ca2+ states. A key feature is that TnC adopts a dumbbell conformation in both the +Ca2+ and -Ca2+ states. More importantly, the data for the -Ca2+ state suggest a long extension of the troponin IT arm, consisting mainly of TnI. Thus, the troponin complex undergoes a large structural change triggered by Ca2+ binding.     

Calcium binding to the low affinity sites in troponin C induces conformational changes in the high affinity domain. A possible route of information transfer in activation of muscle contraction

Residues 89-100 of troponin C (C89-100) and 96-116 of troponin I (I96-116) interact with each other in the troponin complex (Dalgarno, D.C., Grand, R.J.A., Levine, B.A. Moir, A., J.G., Scott, G.M.M., and Perry, S.V. (1982) FEBS Lett. 150, 54-58) and are necessary for the Ca2+ sensitivity of actomyosin ATPase (Syska, H., Wilkinson, J.M., Grand, R.J.A., and Perry, S.V. (1976) Biochem. J. 153, 375-387 and Grabarek, Z., Drabikowski, W., Leavis, P.C., Rosenfeld, S.S., and Gergely, J. (1981) J. Biol. Chem. 256, 13121-13127). We have studied Ca2+-induced changes in the region C89-100 by monitoring the fluorescence of troponin C (TnC) labeled at Cys-98 with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid. Equilibrium titration of the labeled TnC with Ca2+ indicates that the probe is sensitive to binding to both classes of sites in free TnC as well as in its complex with TnI. When Mg2 X TnC is mixed with Ca2+ in a stopped flow apparatus, there is a rapid fluorescence increase related to Ca2+ binding to the unoccupied sites I and II followed by a slower increase (k = 9.9 s-1) that represents Mg2+-Ca2+ exchange at sites III and IV. In the TnC X TnI complex, the fast phase is much larger and the Mg2+-Ca2+ exchange at sites III and IV results in a small decrease rather than an increase in the fluorescence of the probe. The possibility is discussed that the fast change in the environment of Cys-98 upon Ca2+ binding to sites I and II may be instrumental in triggering activation of the thin filament by facilitating a contact between C89-100 and I96-116.     

Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR

We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca(2+)-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca(2+) state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time tau=1.9ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca(2+) state showed that Cys133 existed on a stable segment (tau=4.8ns) held by TnC. Spectra of reconstituted thin filaments (-Ca(2+) state) revealed that slow mobility (tau=45ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca(2+) concentrations.     

Structural and functional studies on Troponin I and Troponin C interactions

Troponin I (TnI) peptides (TnI inhibitory peptide residues 104-115, Ip; TnI regulatory peptide resides 1-30, TnI1-30), recombinant Troponin C (TnC) and Troponin I mutants were used to study the structural and functional relationship between TnI and TnC. Our results reveal that an intact central D/E helix in TnC is required to maintain the ability of TnC to release the TnI inhibition of the acto-S1-TM ATPase activity. Ca(2+)-titration of the TnC-TnI1-30 complex was monitored by circular dichroism. The results show that binding of TnI1-30 to TnC caused a three-folded increase in Ca(2+) affinity in the high affinity sites (III and IV) of TnC. Gel electrophoresis and high performance liquid chromatography (HPLC) studies demonstrate that the sequences of the N- and C-terminal regions of TnI interact in an anti-parallel fashion with the corresponding N- and C-domain of TnC. Our results also indicate that the N- and C-terminal domains of TnI which flank the TnI inhibitory region (residues 104 to 115) play a vital role in modulating the Ca(2+)- sensitive release of the TnI inhibitory region by TnC within the muscle filament. A modified schematic diagram of the TnC/TnI interaction is proposed.