Structure-based rational design of self-inhibitory peptides to disrupt the intermolecular interaction between the troponin subunits C and I in neuropathic pain.

The troponin (Tn) is a ternary complex consisting of three subunits TnC, TnI and TnT; molecular disruption of the Tn complex has been recognized as an attractive strategy against neuropathic pain. Here, a self-inhibitory peptide is stripped from the switch region of TnI interaction interface with TnC, which is considered as a lead molecular entity and then used to generate potential peptide disruptors of TnC-TnI interaction based on a rational molecular design protocol. The region is a helical peptide segment capped by N- and C-terminal disorders. Molecular dynamics simulation and binding free energy analysis suggests that the switch peptide can interact with TnC in a structurally and energetically independent manner. Terminal truncation of the peptide results in a number of potent TnC binders with considerably simplified structure and moderately decreased activity relative to the native switch. We also employ fluorescence polarization assays to substantiate the computational findings; it is found that the rationally designed peptides exhibit moderate or high affinity to TnC with dissociation constants KD at micromolar level.

Changes in the dynamics of the cardiac troponin C molecule explain the effects of Ca2+-sensitizing mutations.

Cardiac troponin C (cTnC) is the regulatory protein that initiates cardiac contraction in response to Ca2+ TnC binding Ca2+ initiates a cascade of protein-protein interactions that begins with the opening of the N-terminal domain of cTnC, followed by cTnC binding the troponin I switch peptide (TnISW). We have evaluated, through isothermal titration calorimetry and molecular-dynamics simulation, the effect of several clinically relevant mutations (A8V, L29Q, A31S, L48Q, Q50R, and C84Y) on the Ca2+ affinity, structural dynamics, and calculated interaction strengths between cTnC and each of Ca2+ and TnISW Surprisingly the Ca2+ affinity measured by isothermal titration calorimetry was only significantly affected by half of these mutations including L48Q, which had a 10-fold higher affinity than WT, and the Q50R and C84Y mutants, each of which had affinities 3-fold higher than wild type. This suggests that Ca2+ affinity of the N-terminal domain of cTnC in isolation is insufficient to explain the pathogenicity of these mutations. Molecular-dynamics simulation was used to evaluate the effects of these mutations on Ca2+ binding, structural dynamics, and TnI interaction independently. Many of the mutations had a pronounced effect on the balance between the open and closed conformations of the TnC molecule, which provides an indirect mechanism for their pathogenic properties. Our data demonstrate that the structural dynamics of the cTnC molecule are key in determining myofilament Ca2+ sensitivity. Our data further suggest that modulation of the structural dynamics is the underlying molecular mechanism for many disease mutations that are far from the regulatory Ca2+-binding site of cTnC.

Interplay of troponin- and Myosin-based pathways of calcium activation in skeletal and cardiac muscle: the use of W7 as an inhibitor of thin filament activation.

To investigate the interplay between the thin and thick filaments during calcium activation in striated muscle, we employed n-(6-aminohexyl) 5-chloro-1-napthalenesulfonamide (W7) as an inhibitor of troponin C and compared its effects with that of the myosin-specific inhibitor, 2,3-butanedione 2-monoxime (BDM). In both skeletal and cardiac fibers, W7 reversibly inhibited ATPase and tension over the full range of calcium activation between pCa 8.0 and 4.5, resulting in reduced calcium sensitivity and cooperativity of ATPase and tension activations. At maximal activation in skeletal fibers, the W7 concentrations for half-maximal inhibition (KI) were 70-80 micro M for ATPase and 20-30 micro M for tension, nearly >200-fold lower than BDM (20 mM and 5-8 mM, respectively). When W7 (50 microM) and BDM (20 mM) were combined in skeletal fibers, the ATPase and tension-pCa curves exhibited lower apparent cooperativity and maxima and higher calcium sensitivity than expected from two independent activation pathways, suggesting that the interplay between the thin and thick filaments varies with the level of activation. Significantly, the inhibition of W7 increased the ATPase/tension ratio during activation in both muscle types. W7 holds much promise as a potent and reversible inhibitor of thin filament-mediated calcium activation of skeletal and cardiac muscle contraction.

Identification of troponin C antagonists from a phage-displayed random peptide library.

Affinity purification of a phage-displayed library, expressing random peptide 12-mers at the N terminus of protein III, has identified 10 distinct novel sequences which bind troponin C specifically. The troponin C-selected peptides yield a consensus binding sequence of (V/L)(D/E)XLKXXLXXLA. Sequence comparison revealed as much as a 62.5% similarity between phiT5, the peptide sequence of the phage clone with the highest level of binding to troponin C, and the N-terminal region of troponin I isoforms. Biotinylated peptides corresponding to library-derived sequences and similar sequences from various isoforms of troponin I were synthesized shown to bind troponin C specifically. Alkaline phosphatase fusion proteins of two of the phage clone sequences bound troponin C specifically, and were specifically competed by both library-derived and native troponin I peptides. Measurement of equilibrium dissociation constants of the peptides by surface plasmon resonance yielded dissociation constants for troponin C as low as 0.43 microM for pT5; in contrast, dissociation constants for calmodulin were greater than 6 microM for all peptides studied. Nondenaturing polyacrylamide gel electrophoresis demonstrated that pT5 formed a stable complex with troponin C in the presence of calcium. We also found that the pT5 peptide inhibited the maximal calcium-activated tension of rabbit psoas muscle fibers.

Inhibition of troponin C production without affecting other muscle protein synthesis by the antisense oligodeoxynucleotide.

The effect of blocking expression of a specific gene with antisense phosphodiester oligodeoxynucleotides on the coordinate regulation of myogenesis was studied. Different regions of both fast and slow troponin C (TnC) mRNAs were targeted for binding of the antisense oligomer. The 5'-cap region of both mRNAs was found to be the most effective target for inhibiting the expression of these genes. Approximately 40%-60% inhibition of expression of a specific isoform of TnC was achieved. However, inhibition of the TnC expression did not appreciably alter the pattern of myogenesis of mouse C2C12 cells. The differentiated murine muscle cells were able to cope with this reduced level of the target gene expression by antisense phosphodiester oligomers. We have also used a phosphorothioate oligomer targeted against a common sequence within the coding region of both fast and slow TnC mRNAs. This oligomer was found to be ineffective in blocking TnC gene expression.