Systematic mapping of regions of human cardiac troponin I involved in binding to cardiac troponin C: N- and C-terminal low affinity contributing regions.
The Spot method of multiple peptide synthesis was used to map in a systematic manner regions of the human cardiac troponin I sequence (hcTnI) involved in interactions with its physiological partner, troponin C (cTnC). Ninety-six 20-mer peptides describing the entire hcTnI sequence were chemically assembled; their reactivity with [125I]cTnC, in the presence of 3 mM Ca2+, enabled the assignment of six sites of interaction (residues 19-32, 45-54, 129-138, 145-164, 161-178 and 191-210). For several sites, a good correlation with literature data was obtained, thus validating this methodological approach. Synthetic peptides, each containing in their sequence an interaction site, were prepared. As assessed by BIACORE, all of them exhibited an affinity for cTnC in the range of 10(-6)-10(-7) M, except for hcTnI [39-58] which showed a nanomolar affinity. This peptide was also able to block the interaction between hcTnI and cTnC. We therefore postulate that despite the existence of multiple cTnC interaction sites on the hcTnI molecule, only that region of hcTnI allows a stabilization of the complex. Residues 19-32 from the N-terminal cardio-specific extension of hcTnI were also found to be involved in interaction with cTnC; residues 19-32 may correspond to the minimal sequence of the extension which could switch between the N- and C-terminal TnC domains, depending on its phosphorylation state. Finally, two Ca(2+)-dependent cTnC binding domains within the C-terminal part of hcTnI (residues 164-178 and 191-210) were also mapped. The latter site may be linked with the cardiac dysfunction observed in stunned myocardium.
Ferrières
G
G
CNRS UMR, Faculté de Pharmacie, Montpellier, France.
Pugnière
M
M
Mani
J C
JC
Villard
S
S
Laprade
M
M
Doutre
P
P
Pau
B
B
Granier
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D016368:Frameshift Mutation; D006801:Humans; D008969:Molecular Sequence Data; D017682:Myocardial Stunning / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D013997:Time Factors; D019209:Troponin C / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019210:Troponin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D010455:Peptides; D019209:Troponin C; D019210:Troponin I