Divalent cadmium ion binding to human cardiac troponin C provides insight into the structural and thermodynamic properties of heavy metal toxicity.

Divalent cadmium (Cd2+), a toxic heavy metal linked to impaired cardiac function, disrupts calcium-dependent signaling by mimicking Ca2+ and binding to EF-hand proteins. Although Cd2+ acts as an isostructural probe for Ca2+ in EF-hand proteins, its thermodynamic behavior in EF-hand motifs remains far less well characterized. Cardiac troponin C (cTnC), an essential Ca2+ sensor in muscle contraction, provides a well-defined EF-hand scaffold for probing Cd2+ interactions. Using isothermal titration calorimetry (ITC), circular dichroism (CD), and inductively coupled plasma optical emission spectroscopy (ICP-OES), we quantified Ca2+ and Cd2+ binding to full-length human cTnC (hcTnC) and its isolated N- and C-domains. CD shows that both metal ions induce similar α-helical structures with subtle differences and that Cd2+ signatures are retained in mixed-metal species. Calcium binding to human cardiac troponin C (hcTnC) displays a single exothermic event corresponding to two Ca2+ ions and an endothermic event corresponding to one additional Ca2+ ion. In contrast, Cd2+ binding exhibits a high-affinity exothermic event for a single Cd2+ ion and a second weaker exotherm representing two additional Cd2+ ions. Domain-specific studies reveal that Cd2+ binding to the C-domain exhibits negative cooperativity. These measurements also show that domain energetics and interdomain coupling shape the thermodynamic signatures observed in the full-length protein. ITC and ICP-OES confirm that Cd2+ can compete with Ca2+ for at least one binding site. Taken together, these spectral and thermodynamic analyses provide a framework for understanding cadmium-induced perturbations in EF-hand metal binding.