C. elegans Troponin I Peptides from Helix 4 and the COOH‐terminus Bind Troponin C in vitro: Novel Implications for the Structural Basis of Troponin's Regulatory Mechanism

Abstract

Troponin is a complex of three proteins (troponin I, troponin C, and troponin T) that work in concert to regulate muscle contraction, in a calcium‐dependent manner. Troponin C (TnC) senses changes in intracellular calcium levels. Troponin I (TnI) is sufficient to stabilize tropomyosin in a position to inhibit actomyosin interactions. Biochemical and biophysical studies demonstrated that reversible binding of calcium to TnC induces large conformational changes of TnI. These conformational changes switch on/off actomyosin interactions. TnC and TnI interact at multiple regions in either a calcium‐dependent or calcium‐independent manner; however, the structure and function of several parts, including the N‐terminal and C‐terminal regions of vertebrate cardiac TnI, have not been solved. We utilized Caenorhabditis elegans TnC (PAT‐10) and TnI (UNC‐27) to identify their interactions in vitro. Our objective was to gain insight into TnI and TnC interactions. We synthesized 38 overlapping 20‐residue peptides, covering the entire TnI (UNC‐27) sequence, and tested their interactions with full‐length TnC. TnI (UNC‐27) peptides were spotted on a nitrocellulose membrane and exposed to TnC (PAT‐10). We found TnC‐binding peptides that correspond to four regions of TnI: helix 1, the inhibitory region, helix 4, and the COOH‐terminal tail. Vertebrate TnI helix 1 binds the C‐lobe of TnC. Vertebrate TnI inhibitory region moves toward TnC in the presence of calcium. Helix 4 or the COOH‐terminal tail of vertebrate TnI has not been recognized as a TnC‐binding region. We hypothesize that TnI helix 4 interacts with the N‐lobe of TnC, in a rocking motion, with its C‐terminal region having enhanced binding to TnC when the TnC N‐lobe binds calcium.