Systematic mapping of regions of human cardiac troponin I involved in binding to cardiac troponin C: N‐ and C‐terminal low affinity contributing regions

Abstract

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 [125 I]cTnC, in the presence of 3 mM Ca 2+ , enabled the assignment of six sites of interaction (residues 19^32, 45^54, 129^138, 145^164, 161^178 and 1912 10). 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 10 361 0 37 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. ß

Defective Dynamic Properties of Human Cardiac Troponin Mutations

“…TnI(G203S) exhibited global changes in T2/T1 relative to wild-type TnI, indicative of higher flexibility in the C-terminal tail. Both the second-actin Tm binding site (regions 173 to 181) 15) and the TnC binding region (regions 161 to 178, 191 to 199) 27) showed dynamic changes, suggesting altered binding to the binding partners. Compared to the data obtained for TnI(G203S), the differences with the wild-type (TnI) are relatively small in the C-terminal tail of TnI(ÁK183) (Fig.…”

Mapping the ADF/Cofilin Binding Site on Monomeric Actin by Competitive Cross-linking and Peptide Array: Evidence for a Second Binding Site on Monomeric Actin

“…28,40,41 Peptide array Eicosamers (20mer) peptides comprising the amino acid sequence of actin were synthesized on a cellulose membrane, as described formerly, with a frameshift of three amino acids. 42,43 Briefly, peptide synthesis was performed automatically from C to N terminus, whereby the carboxy group of the C-terminal amino acid of each peptide forms an ester bond to an OH-group of the cellulose.…”

“…42,44 After incubation with blocking buffer SU-07-250 (Sigma Genosys, Germany) and several washing procedures, the membrane was incubated for 2 h at 37°C with biotinylated cofilin (125 nM-1.25 μM protein in Trisbuffered-saline (TBS)-Tween-Saccharose). Biotinylation was performed with a biotindisulfide-N-hydroxysuccinimide ester (Sigma-Aldrich, Germany) according to the manufacturers' manual.…”

Phosphorylation-dependent Conformational Transition of the Cardiac Specific N-Extension of Troponin I in Cardiac Troponin

“…29 Phosphorylation at Ser23/24 weakens interactions between the cardiac N-extension of cTnI and the N-lobe of cTnC. 25,27,28,30 To further explore structural relationships within the cardiac specific N-extension, we have completed solution NMR studies and bioinformatics analyses on cTnI(1-32), cTnI(1-32) phosphorylated at Ser23/24 (cTnI(1-32)pp), and cTnI(1-32) having Ser23/24 substituted with Asp (cTnI(1-32)DD), a suitable stable mimetic for examining the structural and dynamic consequences of phosphorylation. 27 We determined the structure of the bisphosphorylated form (at Ser23/24) and found evidence for a less structured N-extension in absence of bisphosphorylation.…”