The structure of cardiac troponin C regulatory domain with bound Cd²⁺ reveals a closed conformation and unique ion coordination

The amino-terminal domain of cardiac troponin C (cNTnC) is an essential Ca²⁺ sensor found in cardiomyocytes. It undergoes a conformational change upon Ca²⁺ binding and transduces the signal to the rest of the troponin complex to initiate cardiac muscle contraction. Two classical EF-hand motifs (EF1 and EF2) are present in cNTnC. Under physiological conditions, only EF2 binds Ca²⁺; EF1 is a vestigial site that has lost its function in binding Ca²⁺ owing to amino-acid sequence changes during evolution. Proteins with EF-hand motifs are capable of binding divalent cations other than calcium. Here, the crystal structure of wild-type (WT) human cNTnC in complex with Cd²⁺ is presented. The structure of Cd²⁺-bound cNTnC with the disease-related mutation L29Q, as well as a structure with the residue differences D2N, V28I, L29Q and G30D (NIQD), which have been shown to have functional importance in Ca²⁺ sensing at lower temperatures in ectothermic species, have also been determined. The structures resemble the overall conformation of NMR structures of Ca²⁺-bound cNTnC, but differ significantly from a previous crystal structure of Cd²⁺-bound cNTnC in complex with deoxycholic acid. The subtle structural changes observed in the region near the mutations may play a role in the increased Ca²⁺ affinity. The 1.4 Å resolution WT cNTnC structure, which is the highest resolution structure yet obtained for cardiac troponin C, reveals a Cd²⁺ ion coordinated in the canonical pentagonal bipyramidal geometry in EF2 despite three residues in the loop being disordered. A Cd²⁺ ion found in the vestigial ion-binding site of EF1 is coordinated in a noncanonical `distorted' octahedral geometry. A comparison of the ion coordination observed within EF-hand-containing proteins for which structures have been solved in the presence of Cd²⁺ is presented. A refolded WT cNTnC structure is also presented.