Tyrosine phosphorylation modifies protein kinase C delta-dependent phosphorylation of cardiac troponin I

Our study identifies tyrosine phosphorylation as a novel protein kinase Cdelta (PKCdelta) activation mechanism that modifies PKCdelta-dependent phosphorylation of cardiac troponin I (cTnI), a myofilament regulatory protein. PKCdelta phosphorylates cTnI at Ser23/Ser24 when activated by lipid cofactors; Src phosphorylates PKCdelta at Tyr311 and Tyr332 leading to enhanced PKCdelta autophosphorylation at Thr505 (its activation loop) and PKCdelta-dependent cTnI phosphorylation at both Ser23/Ser24 and Thr144. The Src-dependent acquisition of cTnI-Thr144 kinase activity is abrogated by Y311F or T505A substitutions. Treatment of detergent-extracted single cardiomyocytes with lipid-activated PKCdelta induces depressed tension at submaximum but not maximum [Ca2+] as expected for cTnI-Ser23/Ser24 phosphorylation. Treatment of myocytes with Src-activated PKCdelta leads to depressed maximum tension and cross-bridge kinetics, attributable to a dominant effect of cTnI-Thr144 phosphorylation. Our data implicate PKCdelta-Tyr311/Thr505 phosphorylation as dynamically regulated modifications that alter PKCdelta enzymology and allow for stimulus-specific control of cardiac mechanics during growth factor stimulation and oxidative stress.     

Introduction of negative charge mimicking protein kinase C phosphorylation of cardiac troponin I. Effects on cardiac troponin C

Protein kinase C phosphorylation of cardiac troponin, the Ca(2+)-sensing switch in muscle contraction, is capable of modulating the response of cardiac muscle to a Ca(2+) ion concentration. The N-domain of cardiac troponin I contains two protein kinase C phosphorylation sites. Although the physiological consequences of phosphorylation at Ser(43)/Ser(45) are known, the molecular mechanisms responsible for these functional changes have yet to be established. In this work, NMR was used to identify conformational and dynamic changes in cardiac troponin C upon binding a phosphomimetic troponin I, having Ser(43)/Ser(45) mutated to Asp. Chemical shift perturbation mapping indicated that residues in helix G were most affected. Smaller chemical shift changes were observed in residues located in the Ca(2+)/Mg(2+)-binding loops. Amide hydrogen/deuterium exchange rates in the C-lobe of troponin C were compared in complexes containing either the wild-type or phosphomimetic N-domain of troponin I. In the presence of a phosphomimetic domain, exchange rates in helix G increased, whereas a decrease in exchange rates for residues mapping to Ca(2+)/Mg(2+)-binding loops III and IV was observed. Increased exchange rates are consistent with destabilization of the Thr(129)-Asp(132) helix capping box previously characterized in helix G. The perturbation of helix G and metal binding loops III and IV suggests that phosphorylation alters metal ion affinity and inter-subunit interactions. Our studies support a novel mechanism for protein kinase C signal transduction, emphasizing the importance of C-lobe Ca(2+)/Mg(2+)-dependent troponin interactions.     

Identification of sites phosphorylated in bovine cardiac troponin I and troponin T by protein kinase C and comparative substrate activity of synthetic peptides containing the phosphorylation sites

As an extension of our previous reports that cardiac and skeletal muscle troponin I (Tn-I) and troponin T (Tn-T) are excellent substrates for protein kinase C (PKC) (Katoh, N., Wise, B. C., and Kuo, J. F. (1983) Biochem. J. 209, 189-195; Mazzei, G. J., and Kuo, J. F. (1984) Biochem. J. 218, 361-369), we have now determined that PKC phosphorylated serine 43 (and/or serine 45), serine 78, and threonine 144 in the free Tn-I subunit and threonine 190, threonine 199, and threonine 280 in the free Tn-T subunit of bovine cardiac troponin. PKC appeared to phosphorylate the same sites of the subunits present in the form of the troponin complex, as indicated by the similarity in the two-dimensional phosphopeptide maps. Although some of the phosphorylation sites were shared by other classes of protein kinases, PKC exhibited a distinct substrate specificity. It was also noted that phosphorylated serine and threonine residues in Tn-I and Tn-T had neighboring basic amino acid residues separated by 1 or 2 other residues both at the amino and carboxyl termini, in agreement with the conclusion of House et al. (House, C., Wettenhall, R. E. H., and Kemp, B. E. (1987) J. Biol. Chem. 262, 772-777) based upon their studies on other substrate proteins. Several peptides having sequences around the phosphorylating sites have been synthesized. The phosphorylation experiments indicated that these peptides were substrates for PKC, and their relative substrate activity (determined by the ratios of Vmax/Km) compared with other proteins, in descending order, was Tn-I = Tn-I(134-154) greater than Tn-T much greater than histone H1 greater than Tn-I(33-35) approximately Tn-T(268-284) greater than Tn-T(179-198) approximately Tn-T(191-209). It is suggested that PKC phosphorylation of Tn-I and Tn-T could be biologically significant in terms of possible modifications in interactions among the individual contractile protein components as well as the Ca2+ sensitivity and activity of actomyosin ATPase.     

Identification of threonine 66 as a functionally critical residue of the interleukin-1 receptor-associated kinase

We have mutated a conserved residue of the death domain of the interleukin-1 (IL-1) receptor-associated kinase (IRAK), threonine 66. The substitution of Thr-66 with alanine or glutamate prevented spontaneous activation of NF-kappaB by overexpressed IRAK but enhanced IL-1-induced activation of the factor. Like the kinase-inactivating mutation, K239S, the T66A and T66E mutations interfered with the ability of IRAK to autophosphorylate and facilitated the interactions of IRAK with TRAF6 and with the IL-1 receptor accessory protein, AcP. Wild-type IRAK constructs tagged with fluorescent proteins formed complexes that adopted a punctate distribution in the cytoplasm. The Thr-66 mutations prevented the formation of these complexes. Measurements of fluorescence resonance energy transfer among fluorescent constructs showed that the Thr-66 mutations abolished the capacity of IRAK to dimerize. In contrast, the K239S mutation did not inhibit dimerization of IRAK as evidenced by fluorescence resonance energy transfer measurements, even though microscopy showed that it prevented the formation of punctate complexes. Our results show that Thr-66 plays a crucial role in the ability of IRAK to form homodimers and that its kinase activity regulates its ability to form high molecular weight complexes. These properties in turn determine key aspects of the signaling function of IRAK.     

Protein kinase C phosphorylation of cardiac troponin I or troponin T inhibits Ca2(+)-stimulated actomyosin MgATPase activity.

Effects of troponin phosphorylation on Ca2(+)-stimulated MgATPase activity of bovine cardiac actomyosin were examined. Phosphorylation by protein kinase C of troponin I and troponin T subunits in troponin or troponin-tropomyosin complex resulted in a decreased Ca2(+)-stimulated MgATPase activity in reconstituted actomyosin, and this effect was reversed by subsequent dephosphorylation by protein phosphatase 1. It was further observed that protein kinase C phosphorylation of either troponin I or troponin T subunits led to a similar inhibition of Ca2(+)-stimulated actomyosin MgATPase activity. In all cases, EC50 values (concentrations causing 50% stimulation) for Ca2+ were not appreciably affected by troponin phosphorylation by protein kinase C. Data from phosphorylation site analysis suggests that phosphorylation of threonine 144 in troponin I and possibly threonine 280 or threonine 199 in troponin T might be important for the observed decrease of Ca2(+)-stimulated actomyosin MgATPase. It is suggested that inhibition of actomyosin MgATPase caused by protein kinase C phosphorylation of troponin I and/or troponin T represents a new mechanism that can account for in part the reported negative inotropic effect of phorbol esters on various cardiac preparations.     

Effects of protein kinase C dependent phosphorylation and a familial hypertrophic cardiomyopathy-related mutation of cardiac troponin I on structural transition of troponin C and myofilament activation

In experiments reported here, we compared tension and thin filament Ca(2+) signaling in preparations containing either wild-type cardiac troponin I (cTnI) or a mutant cTnI with an R146G mutation [cTnI(146G)] linked to familial hypertrophic cardiomyopathy. Myofilament function is altered in association with cTnI phosphorylation by protein kinase C (PKC), which is activated in hypertrophy. Whether there are differential effects of PKC phosphorylation on cTnI compared to cTnI(146G) remains unknown. We therefore also studied cTnI and cTnI(146G) with PKC sites mutated to Glu, which mimics phosphorylation. Compared to cTnI controls, binary complexes with either cTnI(146G) or cTnI(43E/45E/144E) had a small effect on Ca(2+)-dependent structural opening of the N-terminal regulatory domain of cTnC as measured using F?rster resonance energy transfer. However, this structural change was significantly reduced in the cTnC-cTnI(43E/45E/144E/146G) complex. Exchange of cTnI in skinned fiber bundles with cTnI(146G) induced enhanced Ca(2+) sensitivity and an elevated resting tension. Exchange of cTnI with cTnI(43E/45E/144E) induced a depression in Ca(2+) sensitivity and maximum tension. However, compared to cTnI(146G), cTnI(43E/45E/144E/146G) had little additional effects on myofilament response to Ca(2+). By comparing activation of tension to the open state of the N-domain of cTnC with variations in the state of cTnI, we were able to provide data supporting the hypothesis that activation of cardiac myofilaments is tightly coupled to the open state of the N-domain of cTnC. Our data also support the hypothesis that pathological effects of phosphorylation are influenced by mutations in cTnI.     

Phosphorylase kinase phosphorylation of skeletal-muscle troponin T.

When [14C]diacylgalactosylglycerol was added to isolated pea or lettuce chloroplasts linolenate synthesis was seen. The desaturation of [14C]linoleate in diacylgalactosylglycerol to [14C]linolenate was stimulated by the addition of a soluble protein fraction containing lipid-exchange activity. Other [14C]acyl lipids were ineffective, except that [14C]phosphatidylcholine in the presence of UDP-galactose and sn-glycerol 3-phosphate could also supply [14C]linoleate for desaturation. These results are consistent with a role of diacylgalactosylglycerol in linolenate synthesis, as indirectly suggested by labelling experiments.     

Identification of the protein kinase C phosphorylation site in neuromodulin

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin [Alexander, K. A., Cimler, B. M., Meier, K. E., & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113], and we have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar Km values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [gamma-32P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32P-labeled tryptic peptide was generated from phosphorylated neuromodulin. The sequence of this peptide was IQASFR. The serine in this peptide corresponds to position 41 of the entire protein, which is adjacent to or contained within the calmodulin binding domain of neuromodulin. A synthetic peptide, QASFRGHITRKKLKGEK, corresponding to the calmodulin binding domain with a few flanking residues, including serine-41, was also phosphorylated by protein kinase C. We conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmodulin to neuromodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ischemic dysfunction in transgenic mice expressing troponin I lacking protein kinase C phosphorylation sites

To determine the in vivo functional significance of troponin I (TnI) protein kinase C (PKC) phosphorylation sites, we created a transgenic mouse expressing mutant TnI, in which PKC phosphorylation sites at serines-43 and -45 were replaced by alanine. When we used high-perfusate calcium as a PKC activator, developed pressures in transgenic (TG) perfused hearts were similar to wild-type (WT) hearts (P = not significant, NS), though there was a 35% and 32% decrease in peak-systolic intracellular calcium (P < 0.01) and diastolic calcium (P < 0.005), respectively. The calcium transient duration was prolonged in the TG mice also (12-27%, ANOVA, P < 0.01). During global ischemia, TG hearts developed ischemic contracture to a greater extent than WT hearts (41 +/- 18 vs. 69 +/- 10 mmHg, perfusate calcium 3.5 mM, P < 0.01). In conclusion, expression of mutant TnI lacking PKC phosphorylation sites results in a marked alteration in the calcium-pressure relationship, and thus susceptibility to ischemic contracture. The reduced intracellular calcium and prolonged calcium transients suggests that a potent feedback mechanism exists between the myofilament and the processes controlling calcium homeostasis.     

Ordered phosphorylation of a duplicated minimal recognition motif for cAMP-dependent protein kinase present in cardiac troponin I.

Cardiac troponin I contains two adjacent serines in sequence after three arginine residues thus making up a minimally duplicated recognition motif for cAMP-dependent protein kinase. In a synthetic peptide, PVRRRSSANY, the two serine residues are phosphorylated sequentially with the intermediate formation of a monophosphorylated species according to the following reaction sequence: Peptide k1----Peptide-P k2----Peptide-P2. The calculated rat constants are: k1 = 0.435.min-1 and k2 = 0.034.min-1. Sequence analyses of the monophosphopeptide and its tryptic fragments show that the predominant monophosphoform carries phosphate at the second serine.     

Agonist-independent phosphorylation of purified cardiac muscarinic cholinergic receptors by protein kinase C

The results of several studies have suggested that muscarinic cholinergic receptors (mAChR) may be regulated by multiple pathways involving phosphorylation of the receptors. Previous studies have demonstrated that chick heart mAChR are phosphorylated by the beta-adrenergic receptor kinase (beta-AR kinase) in an agonist-dependent manner, and it has been suggested that this process may be linked to receptor desensitization. In this work, we present evidence that protein kinase C can phosphorylate the purified, reconstituted chick heart mAChR and can modify the interaction of the receptors with GTP binding proteins (G-proteins) that couple the receptors to effectors. Phosphorylation of the mAChR with protein kinase C occurred to an extent of approximately 5 mol of P/mol of receptor. Neither the rate nor the extent of the protein kinase C mediated phosphorylation of mAChR was agonist-dependent. Under the conditions tested, the initial rate of phosphorylation of the mAChR by protein kinase C was significantly more rapid than that obtained with the beta-AR kinase. At equilibrium, phosphorylation of mAChR by protein kinase C and beta-AR kinase was partially additive. The functional effects of protein kinase C mediated phosphorylation of the mAChR were assessed by comparing the abilities of purified G-proteins (Gi and Go) to reconstitute high-affinity agonist binding to phosphorylated and nonphosphorylated receptors. A significantly larger percentage of the receptors phosphorylated with protein kinase C exhibited G-protein-dependent high-affinity agonist binding, suggesting that phosphorylation of the receptors by protein kinase C modulates receptor function in a positive manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

The phosphorylation sites of troponin T from white skeletal muscle and the effects of interaction with troponin C on their phosphorylation by phosphorylase kinase.

1. The phosphorylation of troponin T from rabbit white sketetal muscle is catalysed by phosphorylase kinase, but not at a significant rate by bovine 3':5'-cyclic AMP-dependent protein kinase. 2. The amino acid sequences adjacent to the three major phosphorylation sites of troponin T were determined. 3. The serine in the N-terminal peptide (Asx,SerP, Glx)Glu-Val-Glu, is that phosphorylated (SerP, phosphoserine) when the troponin complex is isolated. 4. The other two sites of phosphorylation are located in the sequence Ala-Leu-(Ser, SerP)-Met-Gly-Ala-Asn-Tyr(Ser,SerP)Tyr. 5. When troponin T is phosphorylated in the presence of troponin C, the extent of phosphorylation at each site is considerably decreased. 6. CNBr fragments of troponin T are also phosphorylated by phosphorylase kinase, but the rate of phosphorylation at each site in the CNBr fragments is considerably slower than in the native protein. 7. From these studies it is suggested that troponin C interacts with troponin T in the region containing the two closely situated phosphorylation sites.     

The sites of phosphorylation of rabbit cardiac troponin I by adenosine 3'':5''-cyclic monophosphate-dependent protein kinase. Effect of interaction with troponin C.

1. Troponin I prepared from rabbit hearts contains 1.0-1.5 mol of P/mol when isolated by affinity chromatography. Most of the covalently bound phosphate is located in residues 1-48 of the molecule. 2. 3':5'-Cyclic AMP-dependent protein kinase catalyses phosphorylation at serine-20 and serine-146. Serine-20 is more rapidly phosphorylated than serine-146. 3. In troponin I prepared from frozen hearts by affinity chromatography about 0.3-0.5 mol of P/mol is associated with serine-20 and 0.8-1.0 mol of P/mol with other site(s) in residues 1-48 of the molecule. 4. Phosphorylation at serine-20 and servine-146 is not significantly inhibited by troponin C. 5. The mechansim of the interaction of troponin C with cardiac troponin I is discussed in the light of these results.     

Purified cyclic GMP-dependent protein kinase catalyzes the phosphorylation of cardiac troponin inhibitory subunit (TN-1).

Cyclic AMP- and cGMP-dependent protein kinases catalyze the phosphorylation of cardiac troponin inhibitory subunit (TN-I). Unlike many substrates utilized by both kinases, TN-I is rapidly phosphorylated using relatively low concentrations of the cGMP-dependent protein kinase (0.01 to 0.1 micrometer). At low concentrations of cAMP- and cGMP-dependent protein kinases, approximately twice as much total phosphate is incorporated into TN-I using the cAMP-dependent enzyme. At higher enzyme concentrations, 1 mol of phosphate/mol of TN-I is found using either enzyme. Maximal levels of cAMP- and CGMP-dependent protein kinases do not catalyze additive phosphorylation, suggesting that the two enzymes catalyze the phosphorylation of the same site on TN-I. The results support the concept of overlapping substrate specificity for cAMP- and cGMP-dependent protein kinases, but suggest that cardiac troponin contains additional specificity determinants for the cGMP-dependent protein kinase not found in several other protein substrates.     

Disease-causing mutations in cardiac troponin T: identification of a critical tropomyosin-binding region

Fifteen percent of the mutations causing familial hypertrophic cardiomyopathy are in the troponin T gene. Most mutations are clustered between residues 79 and 179, a region known to bind to tropomyosin at the C-terminus near the complex between the N- and C-termini. Nine mutations were introduced into a troponin T fragment, Gly-hcTnT(70-170), that is soluble, alpha-helical, binds to tropomyosin, promotes the binding of tropomyosin to actin, and stabilizes an overlap complex of N-terminal and C-terminal tropomyosin peptides. Mutations between residues 92 and 110 (Arg92Leu, Arg92Gln, Arg92Trp, Arg94Leu, Ala104Val, and Phe110Ile) impair tropomyosin-dependent functions of troponin T. Except for Ala104Val, these mutants bound less strongly to a tropomyosin affinity column and were less able to stabilize the TM overlap complex, effects that were correlated with increased stability of the troponin T, measured using circular dichroism. All were less effective in promoting the binding of tropomyosin to actin. Mutations within residues 92-110 may cause disease because of altered interaction with tropomyosin at the overlap region, critical for cooperative actin binding and regulatory function. A model for a five-chained coiled-coil for troponin T in the tropomyosin overlap complex is presented. Mutations outside the region (Ile79Asn, Delta 160Glu, and Glu163Lys) functioned normally and must cause disease by another mechanism.     

Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T

Protein dephosphorylation by protein phosphatase 1 (PP1), acting in concert with protein kinase C (PKC) and protein kinase A (PKA), is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used in determining dephosphorylation specificities, Ca(2+)-stimulated Mg(2+)ATPase activities, and Ca(2+) sensitivities. In reconstituted troponin (Tn) complex, PP1 displayed distinct substrate specificity in dephosphorylation of TnT preferentially to TnI, in vitro. In situ phosphorylation of cardiomyocytes with calyculin A, a protein phosphatase inhibitor, resulted in an increase in the phosphorylation stiochiometry of TnT (0.3 to 0.5 (67%)), TnI (2.6 to 3.6 (38%)), and MLC2 (0.4 to 1.7 (325%)). These results further confirmed that though MLC2 is the preferred target substrate for protein phosphatase in the thick filament, the Tn complex (TnI and TnT) from thin filament and C-protein in the thick filament are also protein phosphatase substrates. Our in vitro dephosphorylation experiments revealed that while PP1 differentially dephosphorylated within TnT at multiple sites, TnI was uniformly dephosphorylated. Phosphopeptide maps from the in vitro experiments show that TnT phosphopeptides at spots 4A and 4B are much more resistant to PP1 dephosphorylation than other TnT phosphopeptides. Mg(2+)ATPase assays of myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 delineated that while PKC and PKA phosphorylation decreased the Ca(2+)-stimulated Mg(2+)ATPase activities, dephosphorylation antagonistically restored it. PKC and PKA phosphorylation decreased Ca(2+) sensitivity to 3.6 microM and 5.0 microM respectively. However, dephosphorylation restored the Mg(2+)ATPase activity of PKC (99%) and PKA (95%), along with the Ca(2+) sensitivities (3.3 microM and 3.0 microM, respectively).     

Cardiac troponin C-L29Q, related to hypertrophic cardiomyopathy, hinders the transduction of the protein kinase A dependent phosphorylation signal from cardiac troponin I to C

We investigated structural and functional aspects of the first mutation in TNNC1, coding for the calcium-binding subunit (cTnC) of cardiac troponin, which was detected in a patient with hypertrophic cardiomyopathy [ Hoffmann B, Schmidt-Traub H, Perrot A, Osterziel KJ & Gessner R (2001) Hum Mut17, 524]. This mutation leads to a leucine-glutamine exchange at position 29 in the nonfunctional calcium-binding site of cTnC. Interestingly, the mutation is located in a putative interaction site for the nonphosphorylated N-terminal arm of cardiac troponin I (cTnI) [ Finley NL, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Seabrook G, Howarth JW, Rana M, Solaro RJ, Cheung HC et al. (1999) EJB Lett453, 107-112]. According to peptide array experiments, the nonphosphorylated cTnI arm interacts with cTnC around L29. This interaction is almost abolished by L29Q, as observed upon protein kinase A-dependent phosphorylation of cTnI at serine 22 and serine 23 in wild-type troponin. With CD spectroscopy, minor changes are observed in the backbone of Ca2+-free and Ca2+-saturated cTnC upon the L29Q replacement. A small, but significant, reduction in calcium sensitivity was detected upon measuring the Ca2+-dependent actomyosin subfragment 1 (actoS1)-ATPase activity and the sliding velocity of thin filaments. The maximum actoS1-ATPase activity, but not the maximum sliding velocity, was significantly enhanced. In addition, we performed our investigations at different levels of protein kinase A-dependent phosphorylation of cTnI. The in vitro assays mainly showed that the Ca2+ sensitivity of the actoS1-ATPase activity, and the mean sliding velocity of thin filaments, were no longer affected by protein kinase A-dependent phosphorylation of cTnI owing to the L29Q exchange in cTnC. The findings imply a hindered transduction of the phosphorylation signal from cTnI to cTnC.     

Identification of the phosphoserine residue in histone H1 phosphorylated by protein kinase C

The site-specific phosphorylation of bovine histone H1 by protein kinase C was investigated in order to further elucidate the substrate specificity of protein kinase C. Protein kinase C was found to phosphorylate histone H1 to 1 mol per mol. Using N-bromosuccinimide and thrombin digestions, the phosphorylation site was localized to the globular region of the protein, containing residues 71-122. A tryptic peptide containing the phosphorylation site was purified. Modification of the phosphoserine followed by amino acid sequence analysis demonstrated that protein kinase C phosphorylated histone H1 on serine 103. This sequence, Gly97-Thr-Gly-Ala-Ser-Gly-Ser(PO4)-Phe-Lys105, supports the contention that basic amino acid residues C-terminal to the phosphorylation site are sufficient determinants for phosphorylation by protein kinase C.     

The solution structure of a cardiac troponin C-troponin I-troponin T complex shows a somewhat compact troponin C interacting with an extended troponin I-troponin T component

We have investigated the structure of the cTnC-cTnI-cTnT(198-298) calcium-saturated, ternary cardiac troponin complex by small-angle scattering with contrast variation. Shape restoration was also applied to the scattering information resulting from the deuterated cTnC subunit, the unlabeled cTnI-cTnT(198-298) subunits, and the entire complex. The experimental results and modeling indicate that cTnC adopts a partially collapsed conformation, while the cTnI-cTnT(198-298) components have an extended, rod-like structure. Shape restoration applied to the X-ray scattering data and the entire contrast variation series suggest that cTnC and the cTnI-cTnT(198-298) component lie with their long axes roughly parallel to one another with a relatively small surface area for interaction. Our findings indicate that the nature of the interactions between TnC and the TnI-TnT component differs significantly between the cardiac and skeletal isoforms as evidenced by the different degrees of compactness between the cardiac TnC and skeletal TnC in their respective ternary complexes and the fact that the cTnC subunit is not highly intertwined with the other subunits, as observed in the binary complex of the skeletal isoforms [Olah, G. A., and Trewhella, J. (1994) Biochemistry 33, 12800-12806].