The NH 2-Terminal Extension Regulates Molecular Conformation and Function of Cardiac Troponin I

Abstract

The function of troponin I (TnI) is essential to muscle contraction and heart function. Three homologous TnI genes are present in vertebrate species encoding cardiac, fast and slow skeletal muscle TnI isoforms. In addition to the core structure conserved in all TnI isoforms, cardiac TnI (cTnI) has an ∼30 amino acids NH2-terminal extension. This segment is a regulatory structure containing the β-adrenergic dependent protein kinase A (PKA) phosphorylation sites. The NH2-terminal extension of cTnI does not contain binding site for other troponin subunits and is removable by restricted proteolysis to provide functional adaptation in stress conditions. The molecular mechanism for the NH2-terminal modifications to regulate cTnI function is not fully understood. In the present study, we tested a novel hypothesis that the NH2-terminal extension may function through modulating the conformation and activity of other regions of cTnI. Monoclonal antibody epitope analysis and protein binding experiments demonstrated that deletion of the NH2-terminal segment altered epitopic conformation in the middle region of cTnI, similar to the effect of PKA phosphorylation. Such targeted long range conformational effect produced changes in the binding affinity for troponin T and troponin C, with the later in a Ca2+-dependent manner. These data suggest that the NH2-terminal extension of cTnI regulates cardiac muscle contractility through modulating molecular conformation and function of the core structure of cTnI.