Photo-Cross-Linking of Rabbit Skeletal Troponin I Deletion Mutants with Troponin C and Its Thiol Mutants:  The Inhibitory Region Enhances Binding of Troponin I Fragments to Troponin C

Abstract

Contraction of vertebrate striated muscle is regulated by the strong Ca(2+)-dependent interaction between troponin I (TnI) and troponin C (TnC). To critically evaluate this interaction, we generated four recombinant deletion fragments of rabbit fast skeletal TnI: the NH2-terminal fragment (TnI1-94), the NH2 terminus and the inhibitory region (TnI1-120), the inhibitory region and the COOH terminus (TnI96-181), and the COOH-terminal fragment (TnI122-181) containing amino acid residues 1-94, 1-120, 96-181, and 122-181, respectively. Native TnC and seven thiol mutants, containing single cysteine residues in the two globular domains and in the central helix of TnC, e.g., Cys-12, Cys-21, Cys-57, Cys-89, Cys-122, Cys-133, and Cys-158, were labeled with 4-maleimidobenzophenone, and their interaction with the recombinant TnI fragments and the synthetic inhibitory peptide (TnI98-114, residues 98-114) was studied by photo-cross-linking. Extensive cross-linking occurred between various domains of TnC and TnI. The cross-linking patterns (a) showed that both NH2- and COOH-terminal fragments of TnI are accessible to both of the globular domains of TnC, (b) indicated that linkage of the NH2- and COOH-terminal sequences to the inhibitory region of TnI (TnIir) caused marked enhancement of cross-linking with native TnC and all seven thiol mutants, and (c) identified the region in TnC where TnIir binds as that containing residues 98, 133, 158, and 57. Thus, the results suggest that TnI and TnC may adopt flexible and dynamic conformations in which multiple interactions involving various domains of the two polypeptides occur and TnIir acting as a linker facilitates these interactions. The interaction of TnI and its fragments with actin, TnC, and TnT, considered together with the biological activity indicates that residues 96-120 represent a key structural and functional region of TnI. Whereas the NH2-terminal region of TnI stabilizes binding to TnC and TnT, the COOH-terminal region stabilizes TnC and actin binding.

Mapping Subdomains in the C-terminal Region of Troponin I Involved in Its Binding to Troponin C and to Thin Filament

“…Those results complement the results of Farah et al (10) showing that the inhibitory region is involved in the Ca 2ϩ -dependent binding to TnC and that TnI binds TnC in an antiparallel manner: the C-terminal region (regulatory) of TnI interacts with the N terminus (regulatory) of TnC, the C-terminal region (structural) of TnC interacts with the N terminus (structural) of TnI, and the inhibitory region of TnI interacts with both the N-and Cterminal regions of TnC. Support to this model was also given by Sheng et al (15) and by Jha et al (16). McKay et al (19), using a peptide corresponding to residues 115-131 of TnI, showed that this region binds to the hydrophobic pocket of N-domain of TnC (36).…”

“…At this time, little is known about the TnI structure, even though information from low resolution structures is available (12,13) and part of its N-terminal region of TnI (residues 1-47) has been crystallized in a complex with TnC (14). However, the use of deletion mutants (10,15,16) and peptides (11,(17)(18)(19) containing regions of TnI has been shown to be a powerful tool for studying the functions of different regions of this protein and for connecting the functions of TnI with its primary structure.…”

Interaction of the Second Binding Region of Troponin I with the Regulatory Domain of Skeletal Muscle Troponin C as Determined by NMR Spectroscopy

“…However, it was not known exactly which residues of TnI bind to TnC nor where this complex occurs on TnC. Mutation studies (23), cross-linking experiments (17,18,22,24,27), and low angle x-ray diffraction structures (15) have suggested that the exposed TnC hydrophobic pockets are the site of TnC⅐TnI interaction. Interestingly the TnC⅐TnI complex formation was insensitive to calcium unless tropomyosin and other members of the troponin complex were present, although calcium was found to stabilize the isolated TnC⅐TnI complex (6,28).…”

Identification and mutagenesis of a highly conserved domain in troponin T responsible for troponin I binding: Potential role for coiled coil interaction

“…Troponin C (TnC), a member of the EF-hand family of proteins, binds Ca 2ϩ , and relieves TnI inhibition of actin-myosin interaction (see ref. 4 and references therein). Troponin T (TnT) is a structural link between the Tn complex and tropomyosin, and it also increases the cooperativity of actin-tropomyosin binding to myosin (5).…”