Structures of four Ca2+-bound troponin C at 2.0 A resolution: further insights into the Ca2+-switch in the calmodulin superfamily.

BACKGROUND: In contrast to Ca2+4-bound calmodulin (CaM), which has evolved to bind to many target sequences and thus regulate the function of a variety of enzymes, troponin C (TnC) is a bistable switch which controls contraction in striated muscles. The specific target of TnC is troponin I (TnI), the inhibitory subunit of the troponin complex on the thin filaments of muscle. To date, only the crystal structure of Ca2+2-bound TnC (i.e. in the 'off' state) had been determined, which together with the structure of Ca2+4-bound CaM formed the basis for the so-called 'HMJ' model of the conformational changes in TnC upon Ca2+ binding. NMR spectroscopic studies of Ca2+4-bound TnC (i.e. in the 'on' state) have recently been carried out, but the detailed conformational changes that take place upon switching from the off to the on state have not yet been described. RESULTS: We have determined the crystal structures of two forms of expressed rabbit Ca2+4-bound TnC to 2.0 A resolution. The structures show that the conformation of the N-terminal lobe (N lobe) is similar to that predicted by the HMJ model. Our results also reveal, in detail, the residues involved in binding of Ca2+ in the regulatory N lobe of the molecule. We show that the central helix, which links the N and C lobes of TnC, is better stabilized in the Ca2+2-bound than in the Ca2+4-bound state of the molecule. Comparison of the crystal structures of the off and on states of TnC reveals the specific linkages in the molecule that change in the transition from off to on state upon Ca2+-binding. Small sequence differences are also shown to account for large functional differences between CaM and TnC. CONCLUSIONS: The two lobes of TnC are designed to respond to Ca2+-binding quite differently, although the structures with bound Ca2+ are very similar. A small number of differences in the sequences of these two lobes accounts for the fact that the C lobe is stabilized only in the open (Ca2+-bound) state, whereas the N lobe can switch between two stable states. This difference accounts for the Ca2+-dependent and Ca2+-independent interactions of the N and C lobe. The C lobe of TnC is always linked to TnI, whereas the N lobe can maintain its regulatory role - binding strongly to TnI at critical levels of Ca2+ - and in contrast, forming a stable closed conformation in the absence of Ca2+.

Degradation of TN-C component of troponin by trypsin.

The peptide pattern obtained during degradation of TN-C component of troponin by trypsin markedly depends on the concentration of calcium ions. In the presence of calcium higher than 0.1 mM a peptide with a mass of about 16 000 daltons is formed, followed by its splitting into two peptides (8500 and 7500 daltons), which accumulate during further digestion. When calcium is sequestred by EGTA the degradation of TN-C is much faster. The first products of digestion are the 11 000 and 7500 dalton peptides. The former one is degraded to 9700 dalton peptide and, subsequently, both peptides are split to small fragments. In the presence of calcium only three -NH2 groups per molecule of TN-C appear during digestion, whereas in its absence almost all peptide bonds available for trypsin are cleaved. The fragments of TN-C obtained in the presence of calcium are still able to interact with TN-I component as judged by disc-electrophoresis performed in the presence of calcium. During digestion in the presence of calcium only a slight decrease of tyrosyl intrinsic fluorescence occurs and the further decrease after removal of calcium bound to TN-C is fully reversible. Similarly, the content of alpha-helix decreases only slowly during digestion in the presence of calcium. All the results suggest that calcium stabilizes the structure of TN-C molecule, so that trypsin in the presence of calcium releases one small peptide from N-terminal not involved in the first "EF-hand" and the other from the region between pairs 1-2 and 3-4 of "EF-hands" in the nomenclature of Kretsinger (see Kretsinger and Barry [16]). The obtained fragments, containing almost exclusively pairs of "EF-hands", restore most of the properties of original TN-C molecule.

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

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

Digestion of troponin C with trypsin in the presence and absence of Ca2+. Identification of cleavage points.

The rate of tryptic digestion of troponin C has been shown to be dependent on Ca2+ (Drabikowski et al., Biochim. Biophys. Acta 490, 216-224). We have characterized the tryptic peptides produced both in the presence and absence of Ca2+ using amino acid composition and end-group analyses. In the presence of Ca2+ trypsin cleaves TnC at Arg-8, Lys-84 and Lys-88, leading to the formation of two large peptides, one containing the two low-affinity sites (TR1C), the other, the two high-affinity Ca2+-binding sites (TR2C). In the absence of Ca2+ (1 mM EDTA), digestion proceeds much more rapidly and takes place first at Arg-100, followed by Arg-104, Arg-120, Lys-153, Arg-8 and others. The data suggest that the points of cleavage are determined by the Ca2+-dependent conformational states of TnC, particularly in the C-terminal half of the protein where the cation is known to induce secondary structure.

Proton magnetic resonance studies on proteolytic fragments of troponin-C. Structural homology with the native molecule.

Comparison of proton magnetic resonance spectra of a tryptic and a thrombin fragment of troponin-C with that of the native protein has identified the domain of the molecule influenced by Ca2+ binding to the lower affinity regions I and II of troponin-C. The binding of Ca2+ to these sites results in a subtle alteration of the tertiary fold of the N-terminal half of troponin-C involving weakened contacts between several hydrophobic groups. The role and kinetics of the movements within the troponin-C molecule associated with binding at the regulatory sites are discussed.

The nature of the trifluoperazine binding sites on calmodulin and troponin-C.

We have employed 1H-nuclear magnetic resonance spectroscopy to study the interaction of the drug trifluoperazine with calmodulin and troponin-C. Distinct trifluoperazine-binding sites exist in the N- and C-terminal halves of both proteins. Each site consists of a group of hydrophobic side-chains brought into proximity by the Ca2+-dependent juxtaposition of two alpha-helical segments of the protein, each, in turn, belonging to a different Ca2+-binding site in the protein half. The trifluoperazine-induced inhibition of the biological activating ability of calmodulin appears to result from conformational restrictions conferred upon the protein by the bound drug.

Quantitative determination of the content of available methionine and cysteine in food proteins.

The content of available sulphur amino acids in food proteins has been determined by the chemical methods after preliminary digestion of proteins with pancreatin. The values for the available methionine and cysteine contents of pure protein (casein, bovine serum albumin) and protein of food (fresh milk, whey, mackerel, beef, pork, wheat flour) estimated by the specific chemical methods were similar to those for the total content determined by the method of Moore et al. (6). Differences between total and available methionine and cysteine contents of heated casein were found.

The molecular switch in troponin C.

Conformational changes in troponin C (TnC) associated with Ca(2+)-induced triggering of muscle contraction are discussed in light of the model proposed by Herzberg, Moult and James (J. Biol. Chem. 261, 2638, 1986) and of our recent work on mutants of troponin C. The model involves a Ca(2+)-induced angular movement of one pair of alpha-helical segments relative to another pair of helices in the N-terminal domain. A disulfide bridge introduced into the N-terminal domain reversibly blocks the key conformational transition and the Ca(2+)-regulatory activity. Binding of troponin I (TnI) to the disulfide form of TnC is weakened owing to the blocking of its interaction with the N-terminal domain; however incorporation of the mutant into TnC-extracted myofibrils is not abolished. Introduction of a Cys residue in the C-terminal domain of TnC leads to disulfide formation between it and the indigenous Cys-98, with accompanying inhibition of regulatory activity attributable to interference with binding to TnI and, consequently, incorporation into the thin filaments. Evidence for movement of helical segments upon Ca(2+)-binding to TnC was obtained by measurements of excimer fluorescence and of resonance energy transfer with probes attached to Cys residues introduced by site-directed mutagenesis at suitable locations. Introduction of a disulfide bridge into calmodulin, another member of the super-family of Ca(2+)-binding proteins to which TnC belongs, abolishes its interaction with target enzymes. This suggests that the type of conformational change involving angular movement of helical segments that takes place in TnC is also involved in signal transmission in other Ca(2+)-dependent regulatory proteins.

Transmission of the Ca2+-regulatory signal in skeletal muscle thin filaments.

Studies dealing with some aspects of the thin-filament based regulatory mechanism of striated muscle are discussed. A key event is a Ca2+-induced shift of TnI from a binding site on actin to TnC, as indicated by optical and crosslinking experiments. The regions concerned involve residues 1-12 in actin, residues 96-116 in TnI, and residues 89-100 in TnC. These changes in interaction are accompanied by changes in TnC and TnI that occur on a millisecond time scale which is consistent with their being part of the regulatory processes underlying regulation in vivo.

Information transfer in the regulation of striated muscle contraction.

A brief review of current views on the regulation of striated muscle contraction is presented with emphasis on the transmission of the effect of calcium binding to the N-terminal domain of troponin C to the other components of the regulatory system. Results of recent work on chemical modification of troponin C as well as studies on crosslinking among thin filament components are discussed.

Zero-length crosslinking procedure with the use of active esters.

A two-step zero-length crosslinking procedure for studying protein-protein complexes has been developed. One component of a complex is briefly incubated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of N-hydroxysuccinimide resulting in the conversion of some of the protein carboxyls into succinimidyl esters. The reaction is stopped by addition of beta-mercaptoethanol and other interacting proteins are then added. Crosslinking arises from substitution of lysine epsilon-amino groups of these proteins for the succinimidyl moieties during a 1- to 2-h incubation period. The advantage of this method versus one-step zero-length crosslinking is that only one component of the complex is exposed to the crosslinker, which eliminates complications arising from the formation of crosslinks among several proteins of a multicomponent complex. Furthermore, crosslinks can be formed even in the presence of reagents, such as dithiothreitol and EDTA, that would interfere with direct crosslinking with EDC.

Properties of troponin C acetylated at lysine residues.

We have studied the properties of rabbit skeletal troponin C (TnC) fully acetylated at its lysine residues (AcTnC). Acetylation causes a decrease in thermal stability of both domains of TnC in the absence of Ca2+. At 25 degrees C, the acetylated C-terminal domain of TnC is almost completely unfolded and the melting temperature of the N-terminal domain monitored by far-UV circular dichroism is decreased by 16.3 degrees C. In the presence of 1 mM CaCl2, no cooperative unfolding can be detected up to 90 degrees C for either TnC or AcTnC. At 25 degrees C, CD spectra show that AcTnC has a slightly lower alpha-helix content in the absence of Ca2+, but higher in the presence of Ca2+ as compared to unmodified TnC. Acetylation causes a 3.5-fold increase in affinity for Ca2+ at the low-affinity sites and a 2-fold decrease at the high-affinity sites. Polyacrylamide gel electrophoresis under nondissociating conditions (no SDS, no urea, pH 8.6) indicates that acetylation has little effect on the apparent affinity of TnC for troponin I; however, the binding of the acetylated peptides corresponding to the N-terminal domain of TnC to troponin I is significantly stronger than that of the unmodified peptides. Troponin T binding to AcTnC is significantly enhanced, the altered properties of the N-terminal domain being predominantly responsible for the increase. Titration of the ATPase activity of TnC-depleted myofibrils with AcTnC or native TnC indicates that acetylation increases TnC's affinity for myofibrils in the presence of Ca2+ approximately 6 times; at saturation the ATPase activity is the same for the two forms of TnC. The Ca2+ dependence of the ATPase activity of myofibrils containing AcTnC is shifted to lower Ca2+ concentrations, consistent with the higher Ca2+ affinity of AcTnC at the low-affinity sites. These data indicate that positively charged lysine side chains, especially those located in the N-terminal domain, modulate TnC's structural stability and interactions with Ca2+ and other troponin components.

The role of Phe-92 in the Ca(2+)-induced conformational transition in the C-terminal domain of calmodulin.

Recent studies have shown that substitution of Ala for one or more Phe residues in calmodulin (CaM) imparts a temperature-sensitive phenotype to yeast (Ohya, Y., and Botstein, D. (1994) Science 263, 963-966). The Phe residue immediately preceding the first Ca(2+) ligand in site III of CaM (Phe-92) was found to be of particular importance because the mutation at this position alone was sufficient to induce this phenotype. In the present work we have studied the functional and structural consequences of the Phe-92 --> Ala mutation in human liver calmodulin. We found that the mutant (CaMF92A) is incapable of activating phosphodiesterase, and the maximal activation of calcineurin is reduced by 40% as compared with the wild type CaM. Impaired regulatory properties of CaMF92A are accompanied by an increase in affinity for Ca(2+) at the C-terminal domain. To investigate the structural consequences of the F92A mutation, we constructed four recombinant C-terminal domain fragments (C-CaM) of calmodulin (residues 78-148): 1) wild type (C-CaMW); 2) Ala substituted for Phe-92 (C-CaMF92A); 3) cysteine residues introduced at position 85 and 112 to lock the domain with a disulfide bond in the Ca(2+)-free (closed) conformation (C-CaM85/112); and 4) mutations 2 and 3 combined (C-CaM85/112F92A). The Cys-containing mutants readily form intramolecular disulfide bonds regardless whether Phe or Ala is present at position 92. The F92A mutation causes a decrease in stability of the domain in the absence of Ca(2+) as indicated by an 11.8 degree C shift in the far UV circular dichroism thermal unfolding curve. This effect is reversed by the disulfide bond in the C-CaM85/112F92A mutant. The C-CaMW peptide shows a characteristic Ca(2+)-dependent increase in solvent-exposed hydrophobic surface which was monitored by an increase in the fluorescence of the hydrophobic probe 1,1'-bis(4-anilino)-naphthalene-5,5'-disulfonic acid. The fluorescence increase induced by C-CaMF92A is approximately 45% lower than that induced by C-CaMW suggesting that the F92A mutation causes a decrease in the accessibility of several hydrophobic side chains in the C-terminal domain of CaM in the presence of Ca(2+). The Cys-85-Cys-112 disulfide bond causes a 10- or 5.9-fold decrease in Ca(2+) affinity depending on whether Phe or Ala is present at position 92, respectively, suggesting that coupling between Ca(2+) binding and the conformational transition is weaker in the absence of the phenyl ring at position 92. Our results indicate that Phe-92 makes an important contribution to the Ca(2+)-induced transition in the C-terminal domain of CaM. This is most likely the reason for the severely impaired regulatory properties of the CaM mutants having Ala substituted for Phe-92.