Delta 469 mutation in the type 3 repeat calcium binding domain of cartilage oligomeric matrix protein (COMP) disrupts calcium binding.

Cartilage oligomeric matrix protein (COMP/TSP5), a large glycoprotein found in the territorial matrix surrounding chondrocytes, is the fifth member of the thrombospondin (TSP) gene family. While the function of COMP is unknown, its importance is underscored by the finding that mutations in the highly conserved type 3 repeat domain causes two skeletal dysplasias. Pseudoachondroplasia (PSACH) and Multiple Epiphyseal Dysplasia, Fairbanks type (EDM1). The type 3 repeats are highly conserved low-affinity Ca(2+)binding domains that are found in all TSP genes. This study was undertaken to determine the effects of mutations on calcium binding and structure of the type 3 repeat domains. Wild-type (WT) and Delta469 recombinant COMP (rCOMP) proteins containing the entire calcium-binding domain were expressed in E. coli and purified. Equilibrium dialysis demonstrated that WT bound 10-12 Ca(2+)ions/molecule while Delta469 bound approximately half the Ca(2+)ions. Circular dichroism (CD) spectrometry had striking spectral changes for the WT in response to increasing concentrations of Ca(2+). These CD spectral changes were cooperative and reversible. In contrast, a large CD spectral change was not observed at any Ca(2+)concentration for Delta469. Moreover, both WT and Delta469 proteins produced similar CD spectral changes when titrated with Zn(2+), Cu(2+)and Ni(2+)indicating that the Delta469 mutation specifically affects only calcium binding. These results suggest that the Delta469 mutation, in the type 3 repeat region, interferes with Ca(2+)binding and that filling of all Ca(2+)binding loops may be critical for correct COMP protein conformation.

Interaction of cardiac troponin C with Ca(2+) sensitizer EMD 57033 and cardiac troponin I inhibitory peptide.

The binding of Ca(2+) to cardiac troponin C (cTnC) triggers contraction in cardiac muscle. In diseased heart, the myocardium is often desensitized to Ca(2+), leading to weak cardiac contractility. Compounds that can sensitize cardiac muscle to Ca(2+) would have potential therapeutic value in treating heart failure. The thiadiazinone derivative EMD 57033 is an identified 'Ca(2+) sensitizer', and cTnC is a potential target of the drug. In this work, we used 2D ¿(1)H, (15)N¿-HSQC NMR spectroscopy to monitor the binding of EMD 57033 to cTnC in the Ca(2+)-saturated state. By mapping the chemical shift changes to the structure of cTnC, EMD 57033 is found to bind to the C-domain of cTnC. To test whether EMD 57033 competes with cardiac TnI (cTnI) for cTnC and interferes with the inhibitory function, we examined the interaction of cTnC with an inhibitory cTnI peptide (residues 128-147, cIp) in the absence and presence of EMD 57033, respectively. cTnC was also titrated with EMD 57033 in the presence of cIp. The results show that although both the drug and cIp interact with the C-domain of cTnC, they do not displace each other, suggesting noncompetitive binding sites for the two targets. Detailed chemical shift mapping of the binding sites reveals that the regions encompassing helix G-loop IV-helix H are more affected by EMD 57033, while residues located on helix E-loop III-helix F and the linker between sites III and IV are more affected by cIp. In both cases, the binding stoichiometry is 1:1. The binding affinities for the drug are 8.0 +/- 1.8 and 7.4 +/- 4.8 microM in the absence and presence of cIp, respectively, while those for the peptide are 78.2 +/- 10.3 and 99.2 +/- 30.0 microM in the absence and presence of EMD 57033, respectively. These findings suggest that EMD 57033 may exert its positive inotropic effect by not directly enhancing Ca(2+) binding to the Ca(2+) regulatory site of cTnC, but by binding to the structural domain of cTnC, modulating the interaction between cTnC and other thin filament proteins, and increasing the apparent Ca(2+) sensitivity of the contractile system.

Bepridil opens the regulatory N-terminal lobe of cardiac troponin C.

Cardiac troponin C (cTnC) is the calcium-dependent switch for contraction in heart muscle and a potential target for drugs in the therapy of congestive heart failure. This calmodulin-like protein consists of two lobes connected by a central linker; each lobe contains two EF-hand domains. The regulatory N-terminal lobe of cTnC, unlike that of skeletal troponin C (sTnC), contains only one functional EF-hand and does not open fully upon the binding of Ca(2+). We have determined the crystal structure of cTnC, with three bound Ca(2+) ions, complexed with the calcium-sensitizer bepridil, to 2.15-A resolution. In contrast to apo- and 3Ca(2+)-cTnC, the drug-bound complex displays a fully open N-terminal lobe similar to the N-terminal lobes of 4Ca(2+)-sTnC and cTnC bound to a C-terminal fragment of cardiac troponin I (residues 147-163). The closing of the lobe is sterically hindered by one of the three bound bepridils. Our results provide a structural basis for the Ca(2+)-sensitizing effect of bepridil and reveal the details of a distinctive two-stage mechanism for Ca(2+) regulation by troponin C in cardiac muscle.

Drug binding to cardiac troponin C.

Compounds that sensitize cardiac muscle to Ca(2+) by intervening at the level of regulatory thin filament proteins would have potential therapeutic benefit in the treatment of myocardial infarctions. Two putative Ca(2+) sensitizers, EMD 57033 and levosimendan, are reported to bind to cardiac troponin C (cTnC). In this study, we use heteronuclear NMR techniques to study drug binding to [methyl-(13)C]methionine-labeled cTnC when free or when complexed with cardiac troponin I (cTnI). In the absence of Ca(2+), neither drug interacted with cTnC. In the presence of Ca(2+), one molecule of EMD 57033 bound specifically to the C-terminal domain of free cTnC. NMR and equilibrium dialysis failed to demonstrate binding of levosimendan to free cTnC, and the presence of levosimendan had no apparent effect on the Ca(2+) binding affinity of cTnC. Changes in the N-terminal methionine methyl chemical shifts in cTnC upon association with cTnI suggest that cTnI associates with the A-B helical interface and the N terminus of the central helix in cTnC. NMR experiments failed to show evidence of binding of levosimendan to the cTnC.cTnI complex. However, levosimendan covalently bound to a small percentage of free cTnC after prolonged incubation with the protein. These findings suggest that levosimendan exerts its positive inotropic effect by mechanisms that do not involve binding to cTnC.

The kinetic cycle of cardiac troponin C: calcium binding and dissociation at site II trigger slow conformational rearrangements.

The goal of this study is to characterize the kinetic mechanism of Ca2+ activation and inactivation of cardiac troponin C (cTnC), the Ca2+ signaling protein which triggers heart muscle contraction. Previous studies have shown that IAANS covalently coupled to Cys84 of wild-type cTnC is sensitive to conformational change caused by Ca2+ binding to the regulatory site II; the present study also utilizes the C35S mutant, in which Cys84 is the lone cysteine, to ensure the specificity of IAANS labeling. Site II Ca2+ affinities for cTnC-wt, cTnC-C35S, cTnC-wt-IAANS2, and cTnC-C35S-IAANS were similar (KD = 2-5 microM at 25 degrees C; KD = 2-8 microM at 4 degrees C), indicating that neither the IAANS label nor the C35S mutation strongly perturbs site II Ca2+ affinity. To directly determine the rate of Ca2+ dissociation from site II, the Ca2+-loaded protein was rapidly mixed with a spectroscopically sensitive chelator in a stopped flow spectrometer. The resulting site II Ca2+ off-rates were k(off) = 700-800 s(-1) (4 degrees C) for both cTnC-wt and cTnC-C35S, yielding calculated macroscopic site II Ca2+ on-rates of k(on) = k(off)/KD = 2-4 x 10(8) M(-1) s(-1) (4 degrees C). As observed for Ca2+ affinities, neither the C35S mutation nor IAANS labeling significantly altered the Ca2+ on- and off-rates. Using IAANS fluorescence as a monitor of the protein conformational state, the intramolecular conformational changes (delta) induced by Ca2+ binding and release at site II were found to be significantly slower than the Ca2+ on- and off-rates. The conformational rate constants measured for cTnC-wt-IAANS2 and cTnC-C35S-IAANS were k(delta on) = 120-210 s(-1) and k(delta off) = 90-260 s(-1) (4 degrees C) . Both conformational events were slowed in cTnC-wt-IAANS2 relative to cTnC-C35S-IAANS, presumably due to the bulky IAANS probe coupled to Cys35. Together, the results provide a nearly complete kinetic description of the Ca2+ activation cycle of isolated cTnC, revealing rapid Ca2+ binding and release at site II accompanied by slow conformational steps that are likely to be retained by the full troponin complex during heart muscle contraction and relaxation.

A mechanism for calmodulin (CaM) trapping by CaM-kinase II defined by a family of CaM-binding peptides.

Autophosphorylation of Ca2+/calmodulin (CaM)-dependent protein kinase II (CaM-kinase II) induces a striking >1,000-fold increase in its affinity for CaM, which has been called CaM trapping. Two peptides modeled after the CaM binding domain of CaM-kinase II were previously shown to kinetically resemble CaM binding to phosphorylated and dephosphorylated forms of the enzyme, thus providing a model system with which to define the molecular basis of CaM trapping. In this report, the specific contribution of each amino acid to the rates of association and dissociation, and the overall Kd of CaM binding to CaM-kinase II was determined using an overlapping peptide family, and a fluorescently labeled CaM. The association rate constants were similar for the entire family of peptides and ranged from 8 x 10(7) to 32 x 10(7) M-1 s-1. In contrast, the dissociation rate constants for the peptides varied by >3500-fold and ranged from 0.26 to 7 x 10(-5) s-1. These rate constants yield overall Kd values for binding CaM to the peptides that range from 2 x 10(-9) M to 2 x 10(-13) M. Extending the low affinity CaM-binding peptide, CKII(296-312), to include 293Phe-Asn-Ala295 provided the single largest contribution to the decreased dissociation rate constant, 1,300-fold. It was further shown using Ala-substituted peptides that the basic residues 296Arg-Arg-Lys299 were also essential for slow CaM dissociation; however, their contribution was realized only when 293Phe-Asn-Ala295 were present. These results suggest a plausible model in which autophosphorylation of CaM-kinase II leads to a conformational change in the region of 293Phe-Asn-Ala295 which makes these residues accessible for binding to CaM. As a consequence of these changes, further CaM contacts with 296Arg-Arg-Lys299 are established leading to high affinity CaM binding or "CaM trapping."

Identification of binding sites for bepridil and trifluoperazine on cardiac troponin C.

The solution structure of cardiac troponin C (cTnC) (Sia, S., Li, M. X., Spyracopoulos, L., Gagne, S. M., Liu, W., Putkey, J. A. & Sykes, B. D. (1997) J. Biol. Chem. 272, 18216-18221) challenges existing structure/function models for this critical regulatory protein. For example, it is clear that the closed conformation of the regulatory N-terminal domain in Ca2+-bound cardiac troponin C (cTnC) presents a much different binding surface for Ca2+-sensitizing compounds than previously thought. We report here the use of Met methyl groups as site-specific structural markers to identify drug binding sites for trifluoperazine and bepridil on cTnC. Drug dependent changes in the NMR heteronuclear single-quantum coherence spectra of [methyl-13C]Met-labeled cTnC indicate that bepridil and trifluoperazine bind to similar sites but only in the presence of Ca2+. There are 3-4 drug binding sites in the N- and C-terminal domains of intact cTnC that exhibit fast exchange on the NMR time scale. Use of a novel spin-labeled phenothiazine and detection of isotope-filtered nuclear Overhauser effects allowed identification of drug binding sites in the shallow hydrophobic cup in the C-terminal domain and on two hydrophobic surfaces on the N-terminal regulatory domain. The data presented here, coupled with our previous study using covalent blocking groups, support a model in which the Ca2+-sensitizing binding site includes Met-45 in helix B of site I, and Met-60 and -80 in helices B and C of the regulatory site II. This subregion in cTnC makes a likely target against which to design new and selective Ca2+-sensitizing compounds.

The effect of pH on the Ca2+ affinity of the Ca2+ regulatory sites of skeletal and cardiac troponin C in skinned muscle fibres.

It is known that intracellular pH drops rapidly after the onset of ischemia in cardiac muscle and may play some role in the rapid drop in force that ensues. It is also known that alpha 1-adrenoceptor agonists alkalinize intracellular pH by stimulating Na+/H+ exchange and may represent a mechanism which facilitates recovery of intracellular pH from acidosis. Lowering or raising pH shifts the Ca2+ dependence of force development in muscle fibres to higher or lower free Ca2+ concentrations, respectively, yet the precise mechanism is unknown. To investigate this phenomenon we have used skinned skeletal or cardiac muscle fibres whose endogenous troponin C (TnC) has been replaced with chicken skeletal TnC labelled with DANZ (STnCDANZ) or recombinant cardiac TnC labelled with IAANS (CTnC3(C84)[AANS), respectively. The fluorescence of the STnCDANZ or CTnC3(C84)IAANS was enhanced by Ca2+ binding to the Ca(2+)-specific (regulatory) site(s) of STnC or CTnC when incorporated into skinned fibres, and was measured simultaneously with force. When the pH was changed from 7.0 to 6.5 or 7.5 the shift in the Ca2+ dependence of force paralleled the shift in fluorescence. Since the force and fluorescence shift in parallel as the pH is lowered or raised, it can be concluded that these changes in Ca2+ sensitivity are caused by a decrease or increase, respectively, in the Ca2+ affinity of the Ca(2+)-specific site(s) of TnC. Since lowering or raising the pH also resulted in lower or higher, respectively, maximal Ca2+ activated force while maximal fluorescence remained unchanged, it is possible that H+ may act indirectly, as well, by reducing or increasing, respectively, the number or type of crossbridges attached to actin and thereby alter the crossbridge induced depression or elevation, respectively of the observed TnC Ca2+ affinity. Experiments with 2,3-butanedione monoxime, however, where force-generating crossbridges were greatly reduced, indicated that the pH effect may be primarily related to a direct change in the Ca2+ affinity to the regulatory sites of TnC.

Structure of cardiac muscle troponin C unexpectedly reveals a closed regulatory domain.

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.

Fluorescent probes attached to Cys 35 or Cys 84 in cardiac troponin C are differentially sensitive to Ca(2+)-dependent events in vitro and in situ.

The goal of the current study was to generate recombinant cTnC proteins with single Cys residues as sites for attachment of fluorescent probes that can distinguish between the structural effects of myosin cross bridges and direct Ca2+ binding to cTnC (cardiac and slow skeletal troponin C) in skinned fibers. We anticipated that cTnC proteins which retain the endogenous Cys 35 (cTnC(C35)) or Cys 84 (cTnC(C84)) would provide fluorescent probes with distinct microenvironments, since these residues are on opposite sides of the globular regulatory domain. In vitro experiments that showed IAANS (2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid) coupled to Cys 35 can induce unwanted structural perturbations as evidenced by a decreased affinity of site II for Ca2+ when IAANS-labeled cTnC(C35) is bound to cTnI. Important structural features involving Cys 35 in the inactive site I are suggested by a Ca(2+)-dependent increase in reactivity of Cys 35 with sulfhydryl specific reagents when cTnC(C35) is associated with cTnI. These characteristics are not seen for cTnC(C84). When incorporated in situ into skinned cardiac muscle fibers, native cTnC with IAANS bound to both Cys 35 and Cys 84 showed a pCa50 of fluorescence which preceded that of force, while the pCa50 values of both force and fluorescence were coincident for IAANS-labeled cTnC(C84). Disruption of force-producing myosin cross bridges had no effect on the pCa50 of fluorescence for IAANS-labeled cTnC(C84), but induced a rightward shift in the pCa50 of fluorescence for IAANS-labeled native cTnC. These data can be interpreted to indicate that cTnC with IAANS bound to both Cys 35 and C84 senses either myosin cross bridges or direct Ca2+ binding and myosin-induced cooperativity, while IAANS bound to Cys 84 alone senses conformations that are tightly coupled with force generation.

A peptide model for calmodulin trapping by calcium/calmodulin-dependent protein kinase II.

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase) induces a more than 1000-fold increase in calmodulin (CaM)-binding affinity by dramatically decreasing the off-rate for CaM. In this report, we investigate the molecular mechanism for this phenomenon by comparing the rate of dissociation of a novel fluorescently labeled CaM from two synthetic peptides and from the phosphorylated and nonphosphorylated forms of a recombinant preparation of CaM-kinase. Dissociation of a complex of CaM and CKII(296-312), a peptide representing close to the minimum CaM-binding domain of the alpha subunit of CaM-kinase, exhibited a fast off-rate of 5.0 s-1. This was similar to the off-rate of 1.1 s-1 for the dissociation of CaM from the nonphosphorylated form of CaM-kinase. In contrast, dissociation of CaM from either autophosphorylated CaM-kinase or peptide CKII(290-314) was extremely slow with apparent off-rates of about 3-9 x 10(-5) s-1. Along with information from the crystal structure of Ca2+/CaM bound to CKII(290-314) (Meador, W. E., Means, A. R., and Quiocho, F. A. (1993) Science 262, 1718-1721), our results suggest a model in which CaM-dependent autophosphorylation of CaM-kinase induces a conformational change in the region of the CaM-binding domain which allows the formation of additional stabilizing interactions with CaM. We predict that this involves amino acids 293-298 in CaM-kinase. The possible consequences of these observations on the reversibility of CaM trapping in native CaM-kinase are discussed.

Covalent binding of peptides to the N-terminal hydrophobic region of cardiac troponin C has limited effects on function.

Exposure of an N-terminal hydrophobic region in troponin C is thought to be important for the regulation of contraction in striated muscle. To test this hypothesis, single Cys residues were engineered at positions 45, 81, 84, or 85 in the N-terminal hydrophobic region of cardiac troponin C (cTnC) to provide specific sites for attachment of blocking groups. A synthetic peptide, Ac-Val-Arg-Ala-Ile-Gly-Lys-Leu-Ser-Ser, or biotin was coupled to these Cys residues, and the covalent adducts were tested for activity in TnC-extracted myofibrils. Covalent modification of cTnC(C45) had no effect on maximal myofibril ATPase activity. Greatly decreased myofibril ATPase activity (70-80% inhibited) resulted when the peptide was conjugated to Cys-81 in cTnC(C81), while a lesser degree of inhibition (10-25% inhibited) resulted from covalent modification of cTnC(C84) and cTnC(C85). Inhibition was not due to an altered affinity of the cTnC(C81)/peptide conjugate for the myofibrils, and the Ca2+ dependence of ATPase activity was essentially identical to the unmodified protein. Thus, a subregion of the N-terminal hydrophobic region in cTnC is sensitive to disruption, while other regions are less important or can adapt to rather bulky blocking groups. The data suggest that Ca(2+)-sensitizing drugs may bind to the N-terminal hydrophobic region on cTnC but not interfere with transmission of the Ca2+ signal.

An NMR and spin label study of the effects of binding calcium and troponin I inhibitory peptide to cardiac troponin C.

The paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO), was used to probe the surface exposure of methionine residues of recombinant cardiac troponin C (cTnC) in the absence and presence of Ca2+ at the regulatory site (site II), as well as in the presence of the troponin I inhibitory peptide (cTnIp). Methyl resonances of the 10 Met residues of cTnC were chosen as spectral probes because they are thought to play a role in both formation of the N-terminal hydrophobic pocket and in the binding of cTnIp. Proton longitudinal relaxation rates (R1's) of the [13C-methyl] groups in [13C-methyl]Met-labeled cTnC(C35S) were determined using a T1 two-dimensional heteronuclear single- and multiple-quantum coherence pulse sequence. Solvent-exposed Met residues exhibit increased relaxation rates from the paramagnetic effect of HyTEMPO. Relaxation rates in 2Ca(2+)-loaded and Ca(2+)-saturated cTnC, both in the presence and absence of HyTEMPO, permitted the topological mapping of the conformational changes induced by the binding of Ca2+ to site II, the site responsible for triggering muscle contraction. Calcium binding at site II resulted in an increased exposure of Met residues 45 and 81 to the soluble spin label HyTEMPO. This result is consistent with an opening of the hydrophobic pocket in the N-terminal domain of cTnC upon binding Ca2+ at site II. The binding of the inhibitory peptide cTnIp, corresponding to Asn 129 through Ile 149 of cTnI, to both 2Ca(2+)-loaded and Ca(2+)-saturated cTnC was shown to protect Met residues 120 and 157 from HyTEMPO as determined by a decrease in their measured R1 values. These results suggest that in both the 2Ca(2+)-loaded and Ca(2+)-saturated forms of cTnC, cTnIp binds primarily to the C-terminal domain of cTnC.

Assignment and calcium dependence of methionyl epsilon C and epsilon H resonances in cardiac troponin C.

The 10 Met methyl groups in recombinant cardiac troponin (cTnC) were metabolically labeled with [13C-methyl]Met and detected as 10 individual cross-peaks using two-dimensional heteronuclear single- and multiple-quantum coherence (HSMQC) spectroscopy. The epsilon C and epsilon H chemical shifts for all 10 Met residues were sequence-specifically assigned using a combination of HSMQC and systematic conversion of the Met residues to Leu. The only negative functional consequence of these changes was seen when both Met 45 and 81 were mutated. Binding of Ca2+ to the high affinity C-terminal sites III and IV induced relatively large changes in the epsilon H and epsilon C chemical shifts of all Met residues in the C-terminal domain as well as small but significant changes in the chemical shifts of epsilon H Met 47 and Met 81 in the N-terminal half of cTnC. Binding of Ca2+ to the low affinity N-terminal site II induced large changes in the epsilon H and epsilon C chemical shifts of Met 45, Met 80, and Met 81. Binding of Ca2+ to site II had no effect on the chemical shifts of Met residues located in the C-terminal domain. The nature of the chemical shift changes of Met residues in the N- versus the C-terminal halves of cTnC were consistent with different Ca(2+)-induced conformational changes in these domains. Thus, the assigned methyl Met chemical shifts can serve as useful structural markers to study conformational transitional in free cTnC and potentially after association with small ligands, peptides, and other troponin subunits.

The presence but not the sequence of the N-terminal peptide in cardiac TnC is important for function.

The most diverged region of the primary amino acid sequence between cardiac (cTnC) and fast skeletal troponin C is the N-terminal ten amino acids. We report here that major changes in the primary sequence of this region in cTnC had a minimal effect on the ability of the mutant proteins to recover maximal activity in TnC-extracted cardiac and fast skeletal muscle myofibrils. However, deletion of the N-terminal nine amino acids resulted in a 60% decrease in maximal Ca(2+)-dependent ATPase activity with only a small change in the pCa50 of activation. Deletion of the N-terminal peptide did not appear to appreciably affect the Ca(2+)-binding properties of cTnC, but it did alter the interaction with hydrophobic fluorescent probes. Thus, the presence but not the sequence, of the N-terminal extension is important for the maximal activity of cTnC. The N-terminal helix may function in a relatively non-specific manner to prevent unfavorable interactions between domains in cTnC or between cTnC and other troponin subunits.

Differential recovery of Ca2+ binding activity in mutated EF-hands of cardiac troponin C.

Previous studies showed that conversion of the first Ca2+ ligand in Ca(2+)-binding sites III and IV from Asp to Ala decreased the affinity of cardiac TnC (cTnC) for the thin filament. Here, the functional consequences of mutation of the second ligand in the Ca(2+)-binding sites of cTnC were determined. Equilibrium dialysis and Tyr fluorescence studies showed that conversion of the second Ca2+ ligand to Ala (Asp-67, site II; Asn-107, site III; and Asn-143, site IV) inactivated all three Ca(2+)-binding sites in the free protein. Ca2+ binding to the mutated site II was not recovered upon association with a troponin complex, and proteins with this mutation were unable to regulate Ca(2+)-dependent ATPase activity in TnC-extracted myofibrils. However, Ca2+ binding was recovered at the mutated sites III and IV under the same conditions. Sequential addition of active and inactive cTnC proteins in a myofibril ATPase assay suggested that that Mg2+ binding was not recovered and that the recovered Ca2+ affinity of the mutated sites III and IV was much lower than that of the wild type in that the Ca2+ concentrations required for apparent thin filament binding by proteins containing mutations at sites III and/or IV were significantly greater than that required for the wild-type protein.

Calcium plays distinctive structural roles in the N- and C-terminal domains of cardiac troponin C.

One- and two-dimensional NMR techniques were used to compare the structural consequences of Ca2+ binding to both the low and high affinity Ca2+ binding sites in recombinant cardiac troponin C (cTnC3). In the absence of Ca2+, the short beta-sheet located between the high affinity Ca2+/Mg2+ binding sites in the C-terminal domain was found to be absent or loosely formed as judged by the inter-residue NOEs and chemical shifts of resonances in the Ca2+ binding loops. In contrast, the N-terminal domain beta-sheet located between site II and the naturally inactive site I was present even in the absence of bound Ca2+. Calcium-binding mutant proteins having either an inactive Ca2+ binding site III (CBM-III) or an inactive Ca2+ binding site IV (CBM-IV) (Negele, J. C., Dotson, D., Liu, W., Sweeney, H. L., and Putkey, J. A. (1992) J. Biol. Chem. 267, 825-831) were used to study the structural consequences of Ca2+ binding to each of the high affinity sites located in the C-terminal domain. Only a single active Ca2+ binding site was found necessary for formation of the short beta-sheet between Ca2+ binding sites III and IV. However, the absence of bound Ca2+ at site III was found to produce greater instability in the C-terminal domain as judged from the mobility of the C-terminal aromatic hydrophobic cluster. Thus, Ca2+ binding to the high affinity sites in the C-terminal domain results in an ordering of the aromatic hydrophobic cluster, as well as formation of a short beta-sheet between Ca2+ binding sites III and IV. These results demonstrate that Ca2+ binding plays distinctive structural roles in the N- and C-terminal domains of cTnC.

Formation of inter- and intramolecular disulfide bonds can activate cardiac troponin C.

Troponin C regulates contraction in striated muscle by alternating between the Ca(2+)-bound and apo conformations. We report here that spontaneous formation of an intramolecular disulfide bond between Cys-35 and Cys-84, or dimerization via an intermolecular disulfide bond between Cys-84 in cardiac troponin C, renders the protein Ca(2+)-independent when assayed in fast skeletal muscle myofibrils but to a much lesser extent in cardiac myofibrils. Formation of the intramolecular disulfide bond appears to expose hydrophobic surfaces, as indicated by an increase in fluorescence from hydrophobic fluorescent dyes, but does not alter the affinity of Ca(2+)-binding site II. These disulfide bonds constrain the protein into a conformation that either resembles or can substitute for the Ca(2+)-bound form of cardiac troponin C in fast skeletal muscle myofibrils.

Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding.

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.