Ca 2+ and Mg 2+ binding to weak sites of TnC C-domain induces exposure of a large hydrophobic surface that leads to loss of TnC from the thin filament

Abstract

The C-domain of troponin C, the Ca 2+-binding subunit of the troponin complex, has two high-affinity sites for Ca 2+ that also bind Mg 2+ (Ca 2+/Mg 2+ sites), whereas the N-domain has two low-affinity sites for Ca 2+. Two more sites that bind Mg 2+ with very low affinity ( K a < 10 3 M −1) have been detected by several laboratories but have not been localized or studied in any detail. Here we investigated the effects of Ca 2+ and Mg 2+ binding to isolated C-domain, focusing primarily on low-affinity sites. Since TnC has no Trp residues, we utilized a mutant with Phe 154 replaced by Trp (F154W/C-domain). As expected from previous reports, the changes in Trp fluorescence revealed different conformations induced by the addition of Ca 2+ or Mg 2+ (Ca 2+/Mg 2+ sites). Exposure of hydrophobic surfaces of F154W/C-domain was monitored using the fluorescence intensity of bis-anilino naphthalene sulfonic acid. Unlike the changes reported by Trp, the increments in bis-ANS fluorescence were much greater (4.2-fold) when Ca 2+ + Mg 2+ were both present or when Ca 2+ was present at high concentration. Bis-ANS fluorescence increased as a function of [Ca 2+] in two well-defined steps: one at low [Ca 2+], consistent with the Ca 2+/Mg 2+ sites ( K a ∼ 1.5 × 10 6 M −1), and one of much lower affinity ( K a ∼ 52.3 M −1). Controls were performed to rule out artifacts due to aggregation, high ionic strength and formation of the bis-ANS-TnC complex itself. With a low concentration of Ca 2+ (0.6 mM) to occupy the Ca 2+/Mg 2+ sites, a large increase in bis-ANS binding also occurred as Mg 2+ occupied a class of low-affinity sites ( K a ∼ 59 M −1). In skinned fibers, a high concentration of Mg 2+ (10–44 mM) caused TnC to dissociate from the thin filament. These data provide new evidence for a class of weak binding sites for divalent cations. They are located in the C-domain, lead to exposure of a large hydrophobic surface, and destabilize the binding of TnC to the regulatory complex even when sites III and IV are occupied.