Structural details of a calcium-induced molecular switch: X-ray crystallographic analysis of the calcium-saturated N-terminal domain of troponin C at 1.75 A resolution

We have solved and refined the crystal and molecular structures of the calcium-saturated N-terminal domain of troponin C (TnC) to 1.75 A resolution. This has allowed for the first detailed analysis of the calcium binding sites of this molecular switch in the calcium-loaded state. The results provide support for the proposed binding order and qualitatively, for the affinity of calcium in the two regulatory calcium binding sites. Based on a comparison with the high-resolution apo-form of TnC we propose a possible mechanism for the calcium-mediated exposure of a large hydrophobic surface that is central to the initiation of muscle contraction within the cell.     

Tryptophan mutants of troponin C from skeletal muscle--an optical probe of the regulatory domain

We have generated a series of chicken skeletal muscle troponin C mutants to study the conformation of the regulatory domain in the N-terminal half of the molecule. These mutants each contained a single Trp at position 22 (helix A), 52 (linker of helices B and C), or 90 (central helix). Some of these mutants also contained additional mutations to introduce a single Cys at a desired position. The mutants were characterized by molecular graphics and CD and found to have a minimum of structural perturbations when compared with the native structure. They also retained the ability to regulate myofibrillar ATPase activity. The fluorescence of Trp22 was sensitive to Ca2+ binding only to the regulatory sites, whereas Trp52 and Trp90 responded to Ca2+ binding to both the regulatory and the Ca2+/Mg2+ sites. The tryptophan quantum yield (Q) of all Trp22-containing mutants was very high (0.33) in the absence of bound Ca2+, compared to that of L-tryptophan in aqueous solution (0.14). Q decreased 25% upon binding of Ca2+ to the regulatory sites. The quantum yields of Trp52 and Trp90 in apo mutants were close to 0.14. In the presence of bound Ca2+ at the regulatory sites, the quantum yield of Trp52 decreased 16%, whereas that of Trp90 increased 25%. Results from acrylamide quenching of the fluorescence of the three Trp residues indicated that Trp22 was the least exposed and Trp52 was the most exposed, consistent with other spectral data that Trp22 was in a relatively nonpolar environment and Trp52 was in a highly polar environment. The ability of Trp52 and Trp90 to sense Ca2+ binding to sites located at both domains suggests inter-domain communication in the protein. These single Trp TnC mutants provide specific signals for probing Ca2+-induced conformational changes in the regulatory domain.     

Interaction of the second binding region of troponin I with the regulatory domain of skeletal muscle troponin C as determined by NMR spectroscopy

Two dimensional 1H,15N-heteronuclear single quantum correlation NMR was used to monitor the resonance frequency changes of the backbone amide groups belonging to the 15N-labeled regulatory domain of calcium saturated troponin C (N-TnC) upon addition of synthetic skeletal N-acetyl-troponin I 115-131-amide peptide (TnI115-131). Utilizing the change in amide chemical shifts, the dissociation constant for 1:1 binding of TnI115-131 to N-TnC in low salt and 100 mM KCl samples was determined to be 28 +/- 4 and 24 +/- 4 microM, respectively. The off rate of TnI115-131 was determined to be 300 s-1 from observed N-TnC backbone amide 1H,15N-heteronuclear single quantum correlation cross-peak line widths, which is on the order of the calcium off rates (Li, M. X., Gagné, S. M., Tsuda, S., Kay, C. M., Smillie, L. B., and Sykes, B. D. (1995) Biochemistry 34, 8330-8340), and agrees with kinetic expectations for biological regulation of muscle contraction. The TnI115-131 binding site on N-TnC was determined by mapping of chemical shift changes onto the N-TnC NMR structure and was demonstrated to be in the "hydrophobic pocket" (Gagné, S. M., Tsuda, S., Li, M. X., Smillie, L. B., and Sykes, B. D. (1995) Nat. Struct. Biol. 2, 784-789).     

A new method of human cardiac troponin I and troponin T purification

Neurotrophins (NTFs) are a family of structurally related proteins with specific effects on the developing nervous system and a wide range of non-neuronal differentiating cells. To date, four NTFs have been characterized: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). To perform their biological effects, the NTFs must bind to appropriate receptors on the surface of responsive cells. High- and low-affinity receptors for NTFs have been identified. The high-affinity receptors are members of the trk protein tyrosine kinase receptor family. The low-affinity neurotrophin receptor gp75NTFR is a common receptor for all NTFs. Here we summarize some of our previous findings on the expression patterns of NGF, gp75NTFR, TrkB, and TrkC in the developing molar tooth of the rat. Both NGF and gp75NTFR are localized in dental epithelium and mesenchyme but often their expression patterns differ. Concomitant expression of NGF and gp75NTFR in mesenchyme is correlated with odontoblast differentiation. The trkB and trkC receptors show distinct cell-specific expression patterns in developing tooth, suggesting that other NTFs, apart from NGF, may be involved in odontogenesis. These data demonstrate that NTFs participate in the cascade of molecular events that direct tooth development, and support the notion that NTFs may have multiple and distinct roles in dental tissues.     

Backbone and methyl dynamics of the regulatory domain of troponin C: anisotropic rotational diffusion and contribution of conformational entropy to calcium affinity

The N-terminal domain (residues 1 to 90) of chicken skeletal troponin C (NTnC) regulates muscle contraction upon the binding of a calcium ion to each of its two calcium binding loops. In order to characterize the backbone dynamics of NTnC in the apo state (NTnC-apo), we measured and carefully analyzed 15N NMR relaxation parameters T1, T2 and NOE at 1H NMR frequencies of 500 and 600 MHz. The overall rotational correlation time of NTnC-apo at 29.6 degrees C is 4.86 (+/-0.15) ns. The experimental data indicate that the rotational diffusion of NTnC-apo is anisotropic with a diffusion anisotropy, D parallel/D perpendicular, of 1.10. Additionally, the dynamic properties of side-chains having a methyl group were derived from 2H relaxation data of CH2D groups of a partially deuterated sample. Based on the dynamic characteristics of TnC, two different levels of "fine tuning" of the calcium affinity are presented. Significantly lower backbone order parameters (S2), were observed for calcium binding site I relative to site II and the contribution of the bond vector fluctuations to the conformational entropy of sites I and II was calculated. The conformational entropy loss due to calcium binding (DeltaDeltaSp) differs by 1 kcal/mol between sites I and II. This is consistent with the different dissociation constants previously measured for sites I and II of 16 microM and 1. 7 microM, respectively. In addition to the direct role of binding loop dynamics, the side-chain methyl group dynamics play an indirect role through the energetics of the calcium-induced structural change from a closed to an open state. Our results show that the side-chains which will be exposed upon calcium binding have reduced motion in the apo state, suggesting that conformational entropic contributions can be used to offset the free energy cost of exposing hydrophobic groups. It is clear from this work that a complete determination of their dynamic characteristics is necessary in order to fully understand how TnC and other proteins are fine tuned to appropriately carry out their function.     

Clinical utility of cardiac troponin I and cardiac troponin T measurements

The measurement of CK-MB remains the test of choice for confirmation or exclusion of AMI and probably will remain the test of choice for routine diagnosis in the near future. Nowadays determination of cardiac troponin T (cTnT) and cardiac troponin I (cTnI) as a method relatively expensive and time-consuming should be restricted to clinical settings that really require their high specificity.     

Structural determinants of Ca2+ exchange and affinity in the C terminal of cardiac troponin C

The C terminal of cardiac troponin C (TnC) has two Ca2+-Mg2+ sites which exhibit approximately 20-fold higher Ca2+ affinity than the two C-terminal Ca2+ specific sites in calmodulin (CaM). Substitution of the third EF-hand of TnC for the corresponding EF-hand of CaM produced a mutant (CaM[3TnC]) with a 10-fold higher C-terminal Ca2+ and Mg2+ affinity. Substitution of loop 3 of TnC for loop 3 of CaM produced a mutant (CaM[loop3TnC]) with a 10-fold faster Ca2+ on rate and a 5-fold faster Ca2+ off rate than CaM. A mutant CaM (CaM[loop3X, Z]) which contained the identical coordinating amino acids and X and Z acid pairs of TnC loop 3 had a 3-fold higher C-terminal Ca2+ affinity without the increased Ca2+ exchange rates exhibited by CaM[loop3TnC]. Thus, loop factors other than the acid pairs must be responsible for the rapid Ca2+ exchange rates of CaM[loop3TnC]. Helix 6 and helix 5 in the third EF-hand of TnC support the rapid Ca2+ on rate of TnC's loop 3 and produce an approximately 4-fold reduction in its Ca2+ off rate, explaining the high Ca2+ affinity of the third EF-hand of TnC. Exchanging loop 3 or helix 5 of TnC into CaM increased the Mg2+ affinity by decreasing the Mg2+ off rate. Our results are consistent with the high Ca2+ and Mg2+ affinity of the third EF-hand of TnC resulting from the two (X and Z) acid pairs in loop 3, coupled with the greater hydrophobicity of helix 6 and helix 5 compared to that of the third EF-hand of CaM.     

A negative regulatory region in the intracellular domain of the human interferon-alpha receptor

Interferon-alpha (IFN-alpha)-mediated intracellular signaling is initiated by ligand-induced receptor dimerization, tyrosine phosphorylation of the Tyk2 and Jak1 tyrosine kinases, and subsequent phosphorylation of the Stat1 and Stat2 proteins. The IFN-alpha receptor consists of at least two distinct subunits. One subunit, IFNAR1, has low affinity binding for interferon yet is required for signal transduction. We introduced mutations in the cytoplasmic domain of human IFNAR1 in order to identify residues involved in the mediation of biological responses. We took advantage of the species specificity of the interferon receptors by analyzing human IFN-alpha-induced major histocompatibility complex class I antigen expression in mouse L929 cells stably transfected with mutant human receptors. The membrane proximal 60-amino acids were insufficient to signal a biological response even though within these residues Tyk2 and Stat2 binding sites have been identified. IFN-alpha-induced receptor tyrosine phosphorylation was not critical for signaling because mutation of Tyr residues to Phe did not prevent the biological response to IFN-alpha. The deletion of a 16-amino acid region highly homologous between species created a receptor which signals an enhanced response. Tyrosine dephosphorylation is a component of this enhanced response as mutation of the Tyr residues within this region to Phe resulted in a receptor with increased sensitivity to IFN. The known signaling molecules that interact with IFNAR1 are positive regulators of IFN-alpha function. The presence of this domain in the COOH-terminal region suggests that the receptor may interact with signaling molecules that negatively regulate interferon responses.     

Mapping TNNC1, the gene that encodes cardiac troponin I in the human and the mouse

We have mapped the TNNC1 gene, whose protein product is the cardiac TnI protein. TnI is one of the proteins that makes up the troponin complex, which mediates the response of muscle to calcium ions. The human TNNC1 locus had been assigned to a large region of chromosome 19, and we have refined the mapping position to the distal end of the chromosome by amplification of DNAs from a chromosome 19 mapping panel. We have also mapped the mouse Tnnc1 locus, by following the segregation of an intron sequence through DNAs from the European Interspecific Backcross. Tnnc1 maps close to the centromere on mouse chromosome 7.     

Reduced positive feedback regulation between myosin crossbridge and cardiac troponin C in fast skeletal myofibrils

Several studies have shown that substitution of cardiac troponin C into fast skeletal muscle causes a marked reduction in cooperativity of Ca(2+)-activation of both myofibrillar ATPase and tension development. To clarify the underlying mechanisms, in the present study, Ca2+ binding to cardiac troponin C inserted into fast skeletal myofibrils was measured. Two classes of binding sites with different affinities (classes 1 and 2) were clearly identified, which were equivalent stoichiometrically to the two high-affinity sites (sites III and IV) and a single low-affinity site (site II) of troponin C, respectively. Ca2+ binding to class-2 sites and Ca(2+)-activation of myofibrillar ATPase occurred in roughly the same Ca2+ concentration range, indicating that site II is responsible for Ca2+ -regulation. Myosin crossbridge interactions with actin, both in the presence and absence of ATP, enhanced the Ca2+ binding affinity of only class-2 sites. These effects of myosin crossbridges, however, were much smaller than the effects on the Ca2+ binding to the low-affinity sites of fast skeletal troponin C, which are responsible for regulating fast skeletal myofibrillar ATPase. These findings provide strong evidence that the reduction in the cooperative response to Ca2+ upon substituting cardiac troponin C into fast skeletal myofibrils is due to a decrease in the positive feedback interaction between myosin crossbridge attachment and Ca2+ binding to the regulatory site of troponin C.     

The folding kinetics and thermodynamics of the Fyn-SH3 domain

The equilibrium unfolding and the kinetic folding and unfolding of the 67 residue Fyn-SH3 domain have been investigated. Equilibrium unfolding experiments indicate that, despite the lack of both disulfide bonds and prosthetic groups, Fyn-SH3 is relatively stable with a free energy of folding of -6.0 +/- 0.6 kcal mol-1 at 20 degrees C. Kinetic experiments indicate that the domain refolds in a rapid two-state manner without significant population of intermediates (k = 94.3 s-1 in H2O at 20 degrees C). Despite the presence of two proline residues, the refolding of the domain is monophasic, and no significant proline isomerization-like refolding phase is observed. This can be attributed to an extremely low level of the incorrect (cis) isomer of the structurally important Pro134 residue in the protein denatured in 8 M guanidine hydrochloride. Analysis of the temperature and guanidine hydrochloride dependence of the folding rate suggests that the folding transition state of this protein is relatively well organized. A comparison with the refolding kinetics and thermodynamics of other homologous SH3 domains indicates that these exhibit an equivalent degree of transition state organization. This potentially arises from conservation of key features of the transition state conformation despite sometimes relatively low overall sequence identity. Such a comparison further suggests that relative thermodynamic stability is an important factor in determining the relative folding rates of natural proteins with a common fold, but that specific details of the amino acid sequence can also play a significant role in individual cases.     

Photo-cross-linking of rabbit skeletal troponin I deletion mutants with troponin C and its thiol mutants: the inhibitory region enhances binding of troponin I fragments to troponin C

Contraction of vertebrate striated muscle is regulated by the strong Ca(2+)-dependent interaction between troponin I (TnI) and troponin C (TnC). To critically evaluate this interaction, we generated four recombinant deletion fragments of rabbit fast skeletal TnI: the NH2-terminal fragment (TnI1-94), the NH2 terminus and the inhibitory region (TnI1-120), the inhibitory region and the COOH terminus (TnI96-181), and the COOH-terminal fragment (TnI122-181) containing amino acid residues 1-94, 1-120, 96-181, and 122-181, respectively. Native TnC and seven thiol mutants, containing single cysteine residues in the two globular domains and in the central helix of TnC, e.g., Cys-12, Cys-21, Cys-57, Cys-89, Cys-122, Cys-133, and Cys-158, were labeled with 4-maleimidobenzophenone, and their interaction with the recombinant TnI fragments and the synthetic inhibitory peptide (TnI98-114, residues 98-114) was studied by photo-cross-linking. Extensive cross-linking occurred between various domains of TnC and TnI. The cross-linking patterns (a) showed that both NH2- and COOH-terminal fragments of TnI are accessible to both of the globular domains of TnC, (b) indicated that linkage of the NH2- and COOH-terminal sequences to the inhibitory region of TnI (TnIir) caused marked enhancement of cross-linking with native TnC and all seven thiol mutants, and (c) identified the region in TnC where TnIir binds as that containing residues 98, 133, 158, and 57. Thus, the results suggest that TnI and TnC may adopt flexible and dynamic conformations in which multiple interactions involving various domains of the two polypeptides occur and TnIir acting as a linker facilitates these interactions. The interaction of TnI and its fragments with actin, TnC, and TnT, considered together with the biological activity indicates that residues 96-120 represent a key structural and functional region of TnI. Whereas the NH2-terminal region of TnI stabilizes binding to TnC and TnT, the COOH-terminal region stabilizes TnC and actin binding.     

Role of cardiac troponin I in the evaluation of myocardial injury

Congenital fibrosarcoma is a rare soft tissue sarcoma. A 22-year-old woman in the 22nd week of her first pregnancy underwent sonographic examination, which revealed a soft tissue swelling of the fetus's left thigh. The pregnancy was terminated, and congenital fibrosarcoma was diagnosed by pathologic examination. To our knowledge, this is the first published report of the intrauterine sonographic observation of this tumor in a fetal extremity.     

Precolumn affinity capillary electrophoresis for the identification of clinically relevant proteins in human serum: application to human cardiac troponin I

An approach has been developed to the on-line extraction and identification of clinical disease-state marker proteins in human serum. Fabrication of capillaries with integral packed beds for the online determination of human cardiac troponin I (cTnI), a diagnostic marker for myocardial infarction, at clinically relevant levels (2 nmol/L) in serum is demonstrated. The technique, termed precolumn affinity capillary electrophoresis (PA-CE), utilizes a short (approximately 5 mm) packed bed of porous silica containing covalently immobilized monoclonal anti-cTnI antibodies directly integrated within a separation capillary for the selective retention of cTnI from a complex matrix. Following a rinsing step to eliminate nonspecifically bound serum proteins and other impurities from the column, desorption of the antigen into the separation region of the PA-CE capillary for subsequent measurement of femto-molar amounts of cTnI by CE is effected by the injection of an appropriate elution buffer. Advantages of this approach over previously reported affinity preconcentration techniques, related applications for PA-CE technology, and its potential for use in the development of a certified reference material for cTnI in serum are discussed.     

Serum levels of cardiac troponin I and troponin T in estimating myocardial infarct size soon after reperfusion

BACKGROUND: Cardiac troponin I (TnI) and troponin T (TnT) are highly specific myocardial markers. OBJECTIVE: To determine whether their serum levels can be used to estimate myocardial infarct size soon after reperfusion. METHODS: We measured the serum levels of TnI, TnT, and creatine kinase every 3 h, and the serum cardiac myosin light chain I (MLCI) every 24 h, in 42 patients with acute myocardial infarction in whom reperfusion therapy had successfully been performed. We calculated the severity of regional hypokinesis by analyzing the follow-up ventriculograms with the centerline method. RESULTS: The time from reperfusion to the peak level for TnI was 6.1 +/- 3.5 h, significantly shorter than those for creatine kinase (7.5 +/- 4.1 h) and MLCI (55 +/- 28 h). The time to peak level for TnT (6.8 +/- 4.0 h) differed significantly from that for MLCI but not from that for creatine kinase. There was a significant correlation between the peak levels of TnI and TnT (r = 0.86). The peak TnI and TnT levels were correlated well to the peak creatine kinase level (r = 0.67 and 0.69, respectively), total creatine kinase release (r = 0.66 and 0.66), and the peak MLCI level (r = 0.71 and 0.80). We observed excellent correlations between the peak levels of TnI and TnT, and regional hypokinesis (r = -0.84 and -0.85, respectively). These were comparable to the correlations between regional hypokinesis and the peak creatine kinase level (r = 0.75), total creatine kinase release (r = -0.72), and the peak MLCI level (r = -0.76). CONCLUSIONS: These results suggest that the peak serum levels of TnI and TnT in patients with successful reperfusion are accurate and early indices of infarct size.     

Expression of regulated cardiac troponin I in Escherichia coli

The study of the functional effects of troponin isoform changes would be greatly aided by the development of a strategy permitting protein engineering and mutational analysis. To assess the role of troponin isoforms in regulating myofibrillar ATPase activity, we have expressed rat cardiac troponin I (cTnI) in E. coli and purified the protein to near homogeneity. We utilized the inducible expression vector pGEX-KG to create a glutathione-S-transferase fusion protein which can be cleaved with thrombin. Approximately 6 mg of cTnI can be purified from 1 l of culture. Ca2+Mg2+ ATPase activity was measured using the bacterially synthesized cTnI and the remaining components of the regulated actomyosin complex (troponin T, troponin C, tropomyosin, actin, and myosin) purified to homogeneity from mammalian hearts. In the presence of free Ca2+ ranging from 10(-2) to 10(-8) M, bacterially synthesized cTnI exhibits specific activity similar to that observed for control cTnI isolated from rat hearts. The bacterially synthesized protein is capable of stoichiometric phosphorylation and demonstrates appropriately regulated specific activity. These results establish the feasibility of using bacterial expression to study functional consequences of changes in expression of troponin isoforms.     

NMR studies of Ca2+ binding to the regulatory domains of cardiac and E41A skeletal muscle troponin C reveal the importance of site I to energetics of the induced structural changes

Ca2+ binding to the N-domain of skeletal muscle troponin C (sNTnC) induces an "opening" of the structure [Gagné, S. M., et al. (1995) Nat. Struct. Biol. 2, 784-789], which is typical of Ca2+-regulatory proteins. However, the recent structures of the E41A mutant of skeletal troponin C (E41A sNTnC) [Gagné, S. M., et al. (1997) Biochemistry 36, 4386-4392] and of cardiac muscle troponin C (cNTnC) [Sia, S. K., et al. (1997) J. Biol. Chem. 272, 18216-18221] reveal that both of these proteins remain essentially in the "closed" conformation in their Ca2+-saturated states. Both of these proteins are modified in Ca2+-binding site I, albeit differently, suggesting a critical role for this region in the coupling of Ca2+ binding to the induced structural change. To understand the mechanism and the energetics involved in the Ca2+-induced structural transition, Ca2+ binding to E41A sNTnC and to cNTnC have been investigated by using one-dimensional 1H and two-dimensional {1H,15N}-HSQC NMR spectroscopy. Monitoring the chemical shift changes during Ca2+ titration of E41A sNTnC permits us to assign the order of stepwise binding as site II followed by site I and reveals that the mutation reduced the Ca2+ binding affinity of the site I by approximately 100-fold [from KD2 = 16 microM [sNTnC; Li, M. X., et al. (1995) Biochemistry 34, 8330-8340] to 1.3 mM (E41A sNTnC)] and of the site II by approximately 10-fold [from KD1 = 1.7 microM (sNTnC) to 15 microM (E41A sNTnC)]. Ca2+ titration of cNTnC confirms that cNTnC binds only one Ca2+ with a determined dissociation constant KD of 2.6 microM. The Ca2+-induced chemical shift changes occur over the entire sequence in cNTnC, suggesting that the defunct site I is perturbed when site II binds Ca2+. These measurements allow us to dissect the mechanism and energetics of the Ca2+-induced structural changes.