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1.
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. 相似文献
2.
Cardiac troponin C (cTnC) is the Ca(2+)-binding component of the troponin complex and, as such, is the Ca(2+)-dependent switch in muscle contraction. This protein consists of two globular lobes, each containing a pair of EF-hand metal-binding sites, connected by a linker. In the N lobe, Ca(2+)-binding site I is inactive and Ca(2+)-binding site II is primarily responsible for initiation of muscle contraction. The C lobe contains Ca(2+)/Mg(2+)-binding sites III and IV, which bind Mg(2+) with lower affinity and play a structural as well as a secondary role in modulating the Ca(2+) signal. To understand the structural consequences of Ca(2+)/Mg(2+) exchange in the C lobe, we have determined the NMR solution structure of the Mg(2+)-loaded C lobe, cTnC(81-161), in a complex with the N domain of cardiac troponin I, cTnI(33-80), and compared it with a refined Ca(2+)-loaded structure. The overall tertiary structure of the Mg(2+)-loaded C lobe is very similar to that of the refined Ca(2+)-loaded structure as evidenced by the root-mean-square deviation of 0.94 A for all backbone atoms. While metal-dependent conformational changes are minimal, substitution of Mg(2+) for Ca(2+) is characterized by condensation of the C-terminal portion of the metal-binding loops with monodentate Mg(2+) ligation by the conserved Glu at position 12 and partial closure of the cTnI hydrophobic binding cleft around site IV. Thus, conformational plasticity in the Ca(2+)/Mg(2+)-dependent binding loops may represent a mechanism to modulate C-lobe cTnC interactions with the N domain of cTnI. 相似文献
3.
Dong WJ Robinson JM Stagg S Xing J Cheung HC 《The Journal of biological chemistry》2003,278(10):8686-8692
Cardiac muscle activation is initiated by the binding of Ca(2+) to the single N-domain regulatory site of cardiac muscle troponin C (cTnC). Ca(2+) binding causes structural changes between cTnC and two critical regions of cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R, residues 150-165) and the inhibitory region (cTnI-I, residues130-149). These changes are associated with a decreased cTnI affinity for actin and a heightened affinity for cTnC. Using F?rster resonance energy transfer, we have measured three intra-cTnI distances in the deactivated (Mg(2+)-saturated) and Ca(2+)-activated (Ca(2+)-saturated) states in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes. Distance A (spanning cTnI-R) was unaltered by Ca(2+). Distances B (spanning both cTnI-R and cTnI-I) and C (from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca(2+)-induced increases of >8 A. These results compliment our previous determination of the distance between residues flanking cTnI-I alone. Together, the data suggest that Ca(2+) activation causes residues within cTnI-I to switch from a beta-turn/coil to an extended quasi-alpha-helical conformation as the actin-contacts are broken, whereas cTnI-R remains alpha-helical in both Mg(2+)- and Ca(2+)-saturated states. We have used the data to construct a structural model of the cTnI inhibitory and regulatory regions in the Mg(2+)- and Ca(2+)-saturated states. 相似文献
4.
W7 is a well-known calmodulin (CaM) antagonist and has been implicated as an inhibitor of the troponin C-mediated Ca(2+) activation of cardiac muscle contraction. In this study, we use NMR spectroscopy to study binding of W7 to cardiac troponin C (cTnC) free or in complex with cardiac troponin I (cTnI) peptides. Titration of cTnC.3Ca(2+) with W7 shows that residues throughout the sequence, including the N- and C-domains of cTnC and the central linker, are affected. Analysis of the binding stoichiometry and the trajectories of chemical shift changes indicate that W7 binding occurs at multiple sites. To address the issue of whether multiple-site binding is relevant within the troponin complex, W7 is titrated to a cTnC-cTnI complex (cTnC.3Ca(2+).cTnI(34)(-)(71).cTnI(128)(-)(163)). In the presence of the N-terminal (residues approximately 34-71), inhibitory (residues approximately 128-147), and switch (residues approximately 147-163) regions of cTnI, W7 induces chemical shift changes only in the N-domain and not in the C-domain or the central linker of cTnC. The results indicate that in the presence of cTnI, W7 no longer binds to multiple sites of cTnC but instead binds specifically to the N-domain, and the binding (K(D) = 0.5 +/- 0.1 mM) can occur together with the switch region of cTnI. Hence, W7 may play a role in directly modulating the Ca(2+) sensitivity of the regulatory domain of cTnC and the interaction of the switch region of cTnI and cTnC. 相似文献
5.
The binding of W7, an inhibitor of striated muscle contraction, to cardiac troponin C 总被引:1,自引:0,他引:1
W7 is a well-characterized calmodulin antagonist. It decreases the maximal tension and rate of ATP hydrolysis in cardiac muscle fibers. Cardiac troponin C (cTnC) has been previously implicated as the mechanistically significant target for W7 in the myofilament. Two-dimensional NMR spectra ({1H,15N}- and {1H,13C}-HSQCs) were used to monitor the Ca2+-dependent binding of W7 to cTnC. Titration of cTnC x 3Ca2+ with W7 indicated binding to both domains of the protein. We examined the binding of W7 to the separated domains of cTnC to simplify the spectral analysis. In the titration of the C-terminal domain (cCTnC x 2Ca2+), the spectral peaks originating from a subset of residues changed nonuniformly, and could not be well-described as single-site binding. A global fit of the cCTnC x 2Ca2+ titration data to a two-site, sequential binding model (47 residues simultaneously fit) yielded a dissociation constant (Kd1) of 0.85-0.91 mM for the singly bound state, with the second dissociation constant fit to 3.40-3.65 mM (> or = 4 x Kd1). The titration data for the N-terminal domain (cNTnC x Ca2+) was globally fit to single-site binding model with a Kd of 0.15-0.30 mM (41 residues fit). The data are consistent with W7 binding to each domain's major hydrophobic pocket, coordinating side chains responsible for liganding cTnI. When in muscle fibers, W7 may compete with cTnI for target sites on cTnC. 相似文献
6.
W J Dong J Xing M Villain M Hellinger J M Robinson M Chandra R J Solaro P K Umeda H C Cheung 《The Journal of biological chemistry》1999,274(44):31382-31390
Calcium activation of fast striated muscle results from an opening of the regulatory N-terminal domain of fast skeletal troponin C (fsTnC), and a substantial exposure of a hydrophobic patch, essential for Ca(2+)-dependent interaction with fast skeletal troponin I (fsTnI). This interaction is obligatory to relieve the inhibition of strong, force-generating actin-myosin interactions. We have determined intersite distances in the N-terminal domain of cardiac TnC (cTnC) by fluorescence resonance energy transfer measurements and found negligible increases in these distances when the single regulatory site is saturated with Ca(2+). However, in the presence of bound cardiac TnI (cTnI), activator Ca(2+) induces significant increases in the distances and a substantial opening of the N-domain. This open conformation within the cTnC.cTnI complex has properties favorable for the Ca(2+)-induced interaction with an additional segment of cTnI. Thus, the binding of cTnI to cTnC is a prerequisite to achieve a Ca(2+)-induced open N-domain similar to that previously observed in fsTnC with no bound fsTnI. This role of cardiac TnI has not been previously recognized. Our results also indicate that structural information derived from a single protein may not be sufficient for inference of a structure/function relationship. 相似文献
7.
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. 相似文献
8.
An NMR and spin label study of the effects of binding calcium and troponin I inhibitory peptide to cardiac troponin C. 总被引:1,自引:1,他引:0
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J. W. Howarth G. A. Krudy X. Lin J. A. Putkey P. R. Rosevear 《Protein science : a publication of the Protein Society》1995,4(4):671-680
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. 相似文献
9.
The key events in regulating cardiac muscle contraction involve Ca(2+) binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca(2+) movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca(2+) binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca(2+)-induced opening and closing of the N-domain by F?rster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca(2+). L29Q had no effect on the closing rate of the N-domain triggered by release of Ca(2+), but reduced the Ca(2+)-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca(2+) dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC. 相似文献
10.
Wang D Robertson IM Li MX McCully ME Crane ML Luo Z Tu AY Daggett V Sykes BD Regnier M 《Biochemistry》2012,51(22):4473-4487
Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein-protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca(2+) sensitivity of the thin filament have been linked to disease. Tikunova and Davis engineered a series of cNTnC mutations that altered Ca(2+) binding properties and studied the effects on the Ca(2+) sensitivity of the thin filament and contraction [Tikunova, S. B., and Davis, J. P. (2004) J. Biol. Chem. 279, 35341-35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in the cNTnC Ca(2+) binding affinity and Ca(2+) sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca(2+) sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca(2+) and cTnI to cTnC. Nuclear magnetic resonance chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC's conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca(2+) sensitivity can do so by modulating Ca(2+) and cTnI binding. 相似文献
11.
G M Gasmi-Seabrook J W Howarth N Finley E Abusamhadneh V Gaponenko R M Brito R J Solaro P R Rosevear 《Biochemistry》1999,38(26):8313-8322
The N-terminal domain of cardiac troponin I (cTnI) comprising residues 33-80 and lacking the cardiac-specific amino terminus forms a stable binary complex with the C-terminal domain of cardiac troponin C (cTnC) comprising residues 81-161. We have utilized heteronuclear multidimensional NMR to assign the backbone and side-chain resonances of Ca2+-saturated cTnC(81-161) both free and bound to cTnI(33-80). No significant differences were observed between secondary structural elements determined for free and cTnI(33-80)-bound cTnC(81-161). We have determined solution structures of Ca2+-saturated cTnC(81-161) free and bound to cTnI(33-80). While the tertiary structure of cTnC(81-161) is qualitatively similar to that observed free in solution, the binding of cTnI(33-80) results mainly in an opening of the structure and movement of the loop region between helices F and G. Together, these movements provide the binding site for the N-terminal domain of cTnI. The putative binding site for cTnI(33-80) was determined by mapping amide proton and nitrogen chemical shift changes, induced by the binding of cTnI(33-80), onto the C-terminal cTnC structure. The binding interface for cTnI(33-80), as suggested from chemical shift changes, involves predominantly hydrophobic interactions located in the expanded hydrophobic pocket. The largest chemical shift changes were observed in the loop region connecting helices F and G. Inspection of available TnC sequences reveals that these residues are highly conserved, suggesting a common binding motif for the Ca2+/Mg2+-dependent interaction site in the TnC/TnI complex. 相似文献
12.
Gay GL Lindhout DA Sykes BD 《Protein science : a publication of the Protein Society》2004,13(3):640-651
The potential for using paramagnetic lanthanide ions to partially align troponin C in solution as a tool for the structure determination of bound troponin I peptides has been investigated. A prerequisite for these studies is an understanding of the order of lanthanide ion occupancy in the metal binding sites of the protein. Two-dimensional [(1)H, (15)N] HSQC NMR spectroscopy has been used to examine the binding order of Ce(3+), Tb(3+), and Yb(3+) to both apo- and holo-forms of human cardiac troponin C (cTnC) and of Ce(3+) to holo-chicken skeletal troponin C (sTnC). The disappearance of cross-peak resonances in the HSQC spectrum was used to determine the order of occupation of the binding sites in both cTnC and sTnC by each lanthanide. For the lanthanides tested, the binding order follows that of the net charge of the binding site residues from most to least negative; the N-domain calcium binding sites are the first to be filled followed by the C-domain sites. Given this binding order for lanthanide ions, it was demonstrated that it is possible to create a cTnC species with one lanthanide in the N-domain site and two Ca(2+) ions in the C-domain binding sites. By using the species cTnC.Yb(3+).2 Ca(2+) it was possible to confer partial alignment on a bound human cardiac troponin I (cTnI) peptide. Residual dipolar couplings (RDCs) were measured for the resonances in the bound (15)N-labeled cTnI(129-148) by using two-dimensional [(1)H, (15)N] inphase antiphase (IPAP) NMR spectroscopy. 相似文献
13.
Cardiac troponin I (cTnI) is the inhibitory component of the troponin complex, and its interaction with cardiac troponin C (cTnC) plays a critical role in transmitting the Ca(2+) signal to the other myofilament proteins in heart muscle contraction. The switch between contraction and relaxation involves a movement of the inhibitory region of cTnI (cIp) from cTnC to actin-tropomyosin. This region of cTnI is prone to missense mutations in heart disease, and a specific mutation, R145G, has been associated with familial hypertrophic cardiomyopathy. It also contains the unique cardiac PKC phosphorylation site at residue T142. To determine the structural consequences of the mutation R145G and the T142 phosphorylation on the interaction of cIp with cTnC, we have utilized 2D [(1)H, (15)N]-HSQC NMR spectroscopy to monitor the binding of native cIp, cIp-R (R145G), and cIp-P (phosphorylated T142), respectively, to the Ca(2+)-saturated C-domain of cTnC (cCTnC.2Ca(2+)). We also report a strategy for cloning, expression, and purification of cTnI peptide, and both synthetic and recombinant peptides are used in this study. NMR chemical shift mapping indicates that the binding epitope of cIp on cCTnC.2Ca(2+) is not greatly affected, but the affinity is reduced by approximately 14-fold by the T142 phosphorylation and approximately 4-fold by the mutation R145G, respectively. This suggests that these modifications of cIp have an adverse effect on the binding of cIp to cCTnC.2Ca(2+). These perturbations may correlate with the impairment or loss of cTnI function in heart muscle contraction. 相似文献
14.
The interaction of Cardiac Troponin C (cTnC) and Cardiac Troponin I (cTnI) plays a critical role in transmitting the Ca (2+) signal to the other myofilament proteins in the activation of cardiac muscle contraction. As such, the cTnC-cTnI interface is a logical target for cardiotonic agents such as levosimendan that can modulate the Ca (2+) sensitivity of the myofilaments. Evidence indicates that drug candidates may exert their effects by targeting a site formed by binding of the switch region of cTnI to the regulatory N domain of cTnC (cNTnC). In this study, we utilized two-dimensional (1)H- (15)N HSQC NMR spectroscopy to monitor the binding of levosimendan and its analogues, CMDP, AMDP, CI-930, imazodan, and MPDP, to cNTnC.Ca (2+) in complex with two versions of the switch region of cTnI (cTnI 147-163 and cTnI 144-163). Levosimendan, CMDP, AMDP, and CI-930 were found to bind to both cNTnC.Ca (2+).cTnI 147-163 and cNTnC.Ca (2+).cTnI 144-163 complexes. These compounds contain a methyl group that is absent in MPDP or imazodan. Thus, the methyl group is one of the pharmacophores responsible for the action of these pyridazinone drugs on cTnC. Furthermore, the results showed that the cNTnC.Ca (2+).cTnI 144-163 complex presents a higher-affinity binding site for these compounds than the cNTnC.Ca (2+).cTnI 147-163 complex. This is consistent with our observation that the affinity of cTnI 144-163 for cNTnC.Ca (2+) is approximately 10-fold stronger than that of cTnI 147-163, likely a result of electrostatic forces between the N-terminal RRV extension in cTnI 144-163 and the acidic residues in the C and D helices of cNTnC. These results will help in the delineation of the mode of action of levosimendan on the important functional unit of cardiac troponin that constitutes the regulatory domain of cTnC and the switch region of cTnI. 相似文献
15.
An interdomain distance in cardiac troponin C determined by fluorescence spectroscopy 总被引:1,自引:0,他引:1
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Dong WJ Robinson JM Xing J Umeda PK Cheung HC 《Protein science : a publication of the Protein Society》2000,9(2):280-289
The distance between Ca2+-binding site III in the C-terminal domain and Cys35 in the N-terminal domain in cardiac muscle troponin C (cTnC) was determined with a single-tryptophan mutant using bound Tb3+ as the energy donor and iodoacetamidotetramethylrhodamine linked to the cysteine residue as energy acceptor. The luminescence of bound Tb3+ was generated through sensitization by the tryptophan located in the 12-residue binding loop of site III upon irradiation at 295 nm, and this sensitized luminescence was the donor signal transferred to the acceptor. In the absence of bound cation at site II, the mean interdomain distance was found to be 48-49 A regardless of whether the cTnC was unbound or bound to cardiac troponin I, or reconstituted into cardiac troponin. These results suggest that cTnC retains its overall length in the presence of bound target proteins. The distribution of the distances was wide (half-width >9 A) and suggests considerable interdomain flexibility in isolated cTnC, but the distributions became narrower for cTnC in the complexes with the other subunits. In the presence of bound cation at the regulatory site II, the interdomain distance was shortened by 6 A for cTnC, but without an effect on the half-width. The decrease in the mean distance was much smaller or negligible when cTnC was complexed with cTnI or cTnI and cTnT under the same conditions. Although free cTnC has considerable interdomain flexibility, this dynamics is slightly reduced in troponin. These results indicate that the transition from the relaxed state to an activated state in cardiac muscle is not accompanied by a gross alteration of the cTnC conformation in cardiac troponin. 相似文献
16.
Wang X Li MX Spyracopoulos L Beier N Chandra M Solaro RJ Sykes BD 《The Journal of biological chemistry》2001,276(27):25456-25466
Ca(2+) binding to cardiac troponin C (cTnC) triggers contraction in heart muscle. In heart failure, myofilaments response to Ca(2+) are often altered and compounds that sensitize the myofilaments to Ca(2+) possess therapeutic value in this syndrome. One of the most potent and selective Ca(2+) sensitizers is the thiadiazinone derivative EMD 57033, which increases myocardial contractile function both in vivo and in vitro and interacts with cTnC in vitro. We have determined the NMR structure of the 1:1 complex between Ca(2+)-saturated C-domain of human cTnC (cCTnC) and EMD 57033. Favorable hydrophobic interactions between the drug and the protein position EMD 57033 in the hydrophobic cleft of the protein. The drug molecule is orientated such that the chiral group of EMD 57033 fits deep in the hydrophobic pocket and makes several key contacts with the protein. This stereospecific interaction explains why the (-)-enantiomer of EMD 57033 is inactive. Titrations of the cCTnC.EMD 57033 complex with two regions of cardiac troponin I (cTnI(34-71) and cTnI(128-147)) reveal that the drug does not share a common binding epitope with cTnI(128-147) but is completely displaced by cTnI(34-71). These results have important implications for elucidating the mechanism of the Ca(2+) sensitizing effect of EMD 57033 in cardiac muscle contraction. 相似文献
17.
Structure of the regulatory N-domain of human cardiac troponin C in complex with human cardiac troponin I147-163 and bepridil 总被引:2,自引:0,他引:2
Cardiac troponin C (cTnC) is the Ca(2+)-dependent switch for contraction in heart muscle and a potential target for drugs in the therapy of heart failure. Ca(2+) binding to the regulatory domain of cTnC (cNTnC) induces little structural change but sets the stage for cTnI binding. A large "closed" to "open" conformational transition occurs in the regulatory domain upon binding cTnI(147-163) or bepridil. This raises the question of whether cTnI(147-163) and bepridil compete for cNTnC.Ca(2+). In this work, we used two-dimensional (1)H,(15)N-heteronuclear single quantum coherence (HSQC) NMR spectroscopy to examine the binding of bepridil to cNTnC.Ca(2+) in the absence and presence of cTnI(147-163) and of cTnI(147-163) to cNTnC.Ca(2+) in the absence and presence of bepridil. The results show that bepridil and cTnI(147-163) bind cNTnC.Ca(2+) simultaneously but with negative cooperativity. The affinity of cTnI(147-163) for cNTnC.Ca(2+) is reduced approximately 3.5-fold by bepridil and vice versa. Using multinuclear and multidimensional NMR spectroscopy, we have determined the structure of the cNTnC.Ca(2+).cTnI(147-163).bepridil ternary complex. The structure reveals a binding site for cTnI(147-163) primarily located on the A/B interhelical interface and a binding site for bepridil in the hydrophobic pocket of cNTnC.Ca(2+). In the structure, the N terminus of the peptide clashes with part of the bepridil molecule, which explains the negative cooperativity between cTnI(147-163) and bepridil for cNTnC.Ca(2+). This structure provides insights into the features that are important for the design of cTnC-specific cardiotonic drugs, which may be used to modulate the Ca(2+) sensitivity of the myofilaments in heart muscle contraction. 相似文献
18.
Ca(2+) induces an extended conformation of the inhibitory region of troponin I in cardiac muscle troponin. 总被引:1,自引:0,他引:1
The inhibitory region of troponin I (TnI) plays a central regulatory role in the contraction and relaxation cycle of skeletal and cardiac muscle through its Ca(2+)-dependent interaction with actin. Detailed structural information on the interface between TnC and this region of TnI has been long in dispute. We have used fluorescence resonance energy transfer (FRET) to investigate the global conformation of the inhibitory region of a full-length TnI mutant from cardiac muscle (cTnI) in the unbound state and in reconstituted complexes with the other cardiac troponin subunits. The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled with IAEDANS as energy acceptor. The sequence between Trp129 and Cys152 in cTnI brackets the inhibitory region (residues 130-149), and the distance between the two sites was found to be 19.4 A in free cTnI. This distance was insensitive to reconstitution of cTnI with cardiac troponin T (cTnT), cTnC, or cTnC and cTnT in the absence of bound regulatory Ca(2+) in cTnC. An increase of 9 A in the Trp129-Cys152 separation was observed upon saturation of the Ca(2+) regulatory site of cTnC in the complexes. This large increase suggests an extended conformation of the inhibitory region in the interface between cTnC and cTnI in holo cardiac troponin. This extended conformation is different from a recent model of the Ca(2+)-saturated skeletal TnI-TnC complex in which the inhibitory region is modeled as a beta-turn. The observed Ca(2+)-induced conformational change may be a switch mechanism by which movement of the regulatory region of cTnI to the exposed hydrophobic patch of the open regulatory N-domain of cTnC pulls the inhibitory region away from actin upon Ca(2+) activation in cardiac muscle. 相似文献
19.
The Ca(2+)/Mg(2+) sites (III and IV) located in the C-terminal domain of cardiac troponin C (cTnC) have been generally considered to play a purely structural role in keeping the cTnC bound to the thin filament. However, several lines of evidence, including the discovery of cardiomyopathy-associated mutations in the C-domain, have raised the possibility that these sites may have a more complex role in contractile regulation. To explore this possibility, the ATPase activity of rat cardiac myofibrils was assayed under conditions in which no Ca(2+) was bound to the N-terminal regulatory Ca(2+)-binding site (site II). Myosin-S1 was treated with N-ethylmaleimide to create strong-binding myosin heads (NEM-S1), which could activate the cardiac thin filament in the absence of Ca(2+). NEM-S1 activation was assayed at pCa 8.0 to 6.5 and in the presence of either 1mM or 30 μM free Mg(2+). ATPase activity was maximal when sites III and IV were occupied by Mg(2+) and it steadily declined as Ca(2+) displaced Mg(2+). The data suggest that in the absence of Ca(2+) at site II strong-binding myosin crossbridges cause the opening of more active sites on the thin filament if the C-domain is occupied by Mg(2+) rather than Ca(2+). This finding could be relevant to the contraction-relaxation kinetics of cardiac muscle. As Ca(2+) dissociates from site II of cTnC during the early relaxing phase of the cardiac cycle, residual Ca(2+) bound at sites III and IV might facilitate the switching off of the thin filament and the detachment of crossbridges from actin. 相似文献
20.
The Ca(2+) sensitivity of cardiac contractile element is reduced at lower temperatures, in contrast to that in fast skeletal muscle. Cardiac troponin C (cTnC) replacement in mammalian skinned fibers showed that TnC plays a critical role in this phenomenon (Harrison and Bers, (1990), Am. J. Physiol. 258, C282-8). Understanding the differences in affinity and structure between cTnCs from cold-adapted ectothermic species and mammals may bring new insights into how the different isoforms provide different resistances to cold. We followed the Ca(2+) titration to the regulatory domain of rainbow trout cTnC by NMR (wild type at 7 and 30 degrees C and F27W mutant at 30 degrees C) and fluorescence (F27W mutant, at 7 and 30 degrees C) spectroscopies. Using NMR spectroscopy, we detected Ca(2+) binding to site I of trout cTnC at high concentrations. This places trout cTnC between mammalian cTnC, in which site I is completely inactive, and skeletal TnC, in which site I binds Ca(2+) during muscle activation, and which is not as much affected by lower temperatures. This binding was seen both at 7 and at 30 degrees C. Despite the low Ca(2+) affinity, trout TnC site I may increase the likelihood of an opening of the regulatory domain, thus increasing the affinity for TnI. This way, it may be responsible for trout cTnC's capacity to function at lower temperatures. 相似文献