Dynamics and thermodynamics of the regulatory domain of human cardiac troponin C in the apo- and calcium-saturated states.

Abstract

The contraction of cardiac and skeletal muscles is triggered by the binding of Ca2+ to their respective troponin C (TnC) proteins. Recent structural data of both cardiac and skeletal TnC in both the apo and Ca2+ states have revealed that the response to Ca2+ is fundamentally different for these two proteins. For skeletal TnC, binding of two Ca2+ to sites 1 and 2 leads to large changes in the structure, resulting in the exposure of a hydrophobic surface. For cardiac TnC, Ca2+ binds site 2 only, as site 1 is inactive, and the structures show that the Ca2+-induced changes are much smaller and do not result in the exposure of a large hydrophobic surface. To understand the differences between regulation of skeletal and cardiac muscle, we have investigated the effect of Ca2+ binding on the dynamics and thermodynamics of the regulatory N-domain of cardiac TnC (cNTnC) using backbone 15N nuclear magnetic resonance relaxation measurements for comparison to the skeletal system. Analysis of the relaxation data allows for the estimation of the contribution of changes in picosecond to nanosecond time scale motions to the conformational entropy of the Ca2+-binding sites on a per residue basis, which can be related to the structural features of the sites. The results indicate that binding of Ca2+ to the functional site in cNTnC makes the site more rigid with respect to high-frequency motions; this corresponds to a decrease in the conformational entropy (TdeltaS) of the site by 2.2 kcal mol(-1). Although site 1 is defunct, binding to site 2 also decreases the conformational entropy in the nonfunctional site by 0.5 kcal mol(-1). The results indicate that the Ca2+-binding sites in the regulatory domain are structurally and energetically coupled despite the inability of site 1 to bind Ca2+. Comparison between the cardiac and skeletal isoforms in the apo state shows that there is a decrease in conformational entropy of 0.9 kcal mol(-1) for site 1 of cNTnC and little difference for site 2.