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.
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.
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+.
Background: In contrast to Ca
2+
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 Ca
2+
2
-bound TnC (i.e. in the ‘off’ state) had been determined, which together with the structure of Ca
2+
4-bound CaM formed the basis for the so-called ‘HMJ’ model of the conformational changes in TnC upon Ca
2+ binding. NMR spectroscopic studies of Ca
2+
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 Ca
2+
4-bound TnC to 2.0 Å 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 Ca
2+ 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 Ca
2+
2-bound than in the Ca
2+
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 Ca
2+-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 Ca
2+-binding quite differently, although the structures with bound Ca
2+ 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 (Ca
2+-bound) state, whereas the N lobe can switch between two stable states. This difference accounts for the Ca
2+-dependent and Ca
2+-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 Ca
2+ – and in contrast, forming a stable closed conformation in the absence of Ca
2+.
The regulatory activity of troponin C is reversibly inhibited by a disulfide bridge between cysteine residues introduced by
site-directed mutagenesis in positions 48 and 82 (TnC48/82) in the ...N-terminal domain of rabbit skeletal troponin C (sTnC;
Grabarek, Z., Tan, R.-Y., Tao, T., and Gergely, J. (1990) Nature 345, 132-135). In the present work we have investigated the
effects of the disulfide on structural properties of TnC48/82 monitored by CD spectroscopy and limited trypsinolysis. The
CD spectra of the mutant protein in the oxidized form (oxTnC48/82) with and without Ca2+ are similar to the corresponding
ones of the reduced and carboxamidomethylated form (CAMTnC48/82), indicating that the disulfide has essentially no effect
on the overall secondary structure. The N-terminal domain of oxTnC48/82 is resistant to thermal unfolding, but that of CAMTnC48/82
is only slightly more stable than the corresponding domain of sTnC. In the presence of Ca2+ oxTnC48/82 is more resistant to
trypsinolysis than sTnC whereas the rate of tryptic digestion of CAMTnC48/82 is the same as that of sTnC, indicating that
peptide bonds adjacent to lysine residues at position 84 and 88, the sites of tryptic attack, are protected by the disulfide.
The disulfide cross-linked N-terminal peptide of TnC48/82 does not bind TnI, unlike its reduced or carboxamidomethylated forms.
Our data indicate that the disulfide between Cys48 and Cys82 stabilizes the structure of the N-terminal domain of TnC and
blocks its ability to interact with TnI. The effects of the disulfide appear to be restricted to the N-terminal domain of
TnC.
The molecular switch in troponin C Gergely, J; Grabarek, Z; Tao, T
Advances in experimental medicine and biology,
1993, Letnik:
332
Journal Article
Recenzirano
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.
To obtain additional information about the effect of Ca super(2+) and Mg super(2+) on the secondary structure of Ca super(2+)-binding proteins, the authors studied thermostability of skeletal muscle ...troponin C and calmodulin using circular dichroism technique and the influence of Ca super(2+)-and Mg super(2+)-binding on thermal unfolding. Tryptic fragments of both calcium-binding proteins were used to localise the structural changes caused by temperature changes in the particular parts of both molecules.
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