Regulatory Domain Conformational Exchange and Linker Region Flexibility in Cardiac Troponin C Bound to Cardiac Troponin I

M.Bret Abbott,Vadim Gaponenko,Ekram Abusamhadneh,Natosha Finley,Ge Li,Alex Dvoretsky,Mark Rance,R.John Solaro,Paul R Rosevear

doi:10.1074/jbc.m909252199

Abstract

Previously, we utilized (15)N transverse relaxation rates to demonstrate significant mobility in the linker region and conformational exchange in the regulatory domain of Ca(2+)-saturated cardiac troponin C bound to the isolated N-domain of cardiac troponin I (Gaponenko, V., Abusamhadneh, E., Abbott, M. B., Finley, N., Gasmi-Seabrook, G., Solaro, R.J., Rance, M., and Rosevear, P.R. (1999) J. Biol. Chem. 274, 16681-16684). Here we show a large decrease in cardiac troponin C linker flexibility, corresponding to residues 85-93, when bound to intact cardiac troponin I. The addition of 2 m urea to the intact cardiac troponin I-troponin C complex significantly increased linker flexibility. Conformational changes in the regulatory domain of cardiac troponin C were monitored in complexes with troponin I-(1-211), troponin I-(33-211), troponin I-(1-80) and bisphosphorylated troponin I-(1-80). The cardiac specific N terminus, residues 1-32, and the C-domain, residues 81-211, of troponin I are both capable of inducing conformational changes in the troponin C regulatory domain. Phosphorylation of the cardiac specific N terminus reversed its effects on the regulatory domain. These studies provide the first evidence that the cardiac specific N terminus can modulate the function of troponin C by altering the conformational equilibrium of the regulatory domain.

Highlights

We utilized 15N transverse relaxation rates to demonstrate significant mobility in the linker region and conformational exchange in the regulatory domain of Ca2؉-saturated cardiac troponin C bound to the isolated N-domain of cardiac troponin I
The absence of the cardiac specific N terminus (cTnI-(33–211)), mutation of Ser23 and Ser24 to Asp (cTnI-(1– 80)DD) or phosphorylation at Ser residues 23 and 24 (cTnI-(1– 80)pp), shifts the conformational equilibrium toward that observed in free Ca2ϩ-saturated cTnC. This provides the first evidence that the cardiac specific N terminus modulates the function of cTnC by altering the conformational equilibria within the regulatory domain. These results demonstrate that a simple model based on Ca2ϩ-dependent binding of cTnI residues 147–163 to the regulatory domain of cTnC is insufficient to account for the molecular details of cardiac troponin I-troponin C interactions
Investigation of proposed mechanistic roles of the linker region of cTnC and the cardiac specific N terminus of cTnI require a model system based on the intact cTnC molecule

Summary

Introduction

We utilized 15N transverse relaxation rates to demonstrate significant mobility in the linker region and conformational exchange in the regulatory domain of Ca2؉-saturated cardiac troponin C bound to the isolated N-domain of cardiac troponin I Calcium binding to sTnC was suggested to change the angle between TnI domains, whereas the radius of gyration for TnC was not affected (17) Both sets of data are consistent with an extended sTnC structure, the mass distribution for TnI in the in situ complex differs significantly from that found in the TnIC complex in the presence of 2–3 M urea (15–17). High resolution solution NMR studies of TnC and TnI interactions have largely utilized isolated domains and synthetic peptides due to the large molecular mass (approximately 42 kDa) and limited solubility of the intact cTnIC complex. Findings from these studies, by their nature, fail to reveal structural mechanisms that rely on interdomain interactions and must be verified in more sophisticated model systems. They do not contain sufficient detail to verify the findings of the high resolution single-domain experiments or completely elucidate the structural mechanisms of cTnC regulation and cTnI inhibitory function

Methods

Results

Conclusion