Troponin C contains a 14-residue α-helix at the amino terminus, the N- helix, that calmodulin lacks. Deletion of the first 11-14 residues of troponin C alters function. In the present investigation a mutant lacking residues 1-7 of the N-helix has normal conformation, Ca2+ binding, and regulatory function. Thus, residues 8-14 of the N-helix are generally sufficient for troponin C function. In the x-ray structures of troponin C there is a salt bridge between Arg 11 in the N-helix and Glu 76 in the D- helix. Destroying the salt bridge by individually mutating the residues to Cys has no effect on function. However, mutation of both residues to Cys reduces troponin C's affinity for the troponin complex on the thin filament, reduces the stability of the N-domain in the absence of divalent cations, increases the Ca2+ affinity and reduces the cooperativity of the Ca2+Mg2+ sites in the C-domain, and alters the conformational change that takes place upon Ca2+ binding (but not Mg2+ binding) to the C-domain. Cross-linking with bis-(maleimidomethylether) partially restores function. The Ca2+-specific sites in the N-domain, those closest to the sites of the mutations, are unaffected in the assays employed. These results show that the N-helix is a critical structural element for interaction with and activation of the thin filament. Moreover, mutations in the N-helix affect the C- terminal domain, consistent with recent structural studies showing that the N-helix and C-terminal domain are physically close.
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