Thermodynamic and Structural Characterization of Cd(II) binding to human cardiac troponin C: Using Mutants and Truncated Constructs to Elucidate Cd(II) binding sites

Abstract

Many biological processes rely on the presence of essential metals; therefore, maintaining the concentration of metals is paramount to homeostasis. Cadmium, Cd(II), is a toxic heavy metal that can disturb this delicate balance, leading to biochemical disruption through its ability to mimic essential metals. The purpose of this work was to understand Cd(II) binding to human cardiac troponin C (hcTnC), a native Ca(II)-binding protein, at both the thermodynamic and structural levels. In this study, ITC was utilized to determine the thermodynamic parameters for Ca(II) and Cd(II) binding to full length wild type and mutant (C35A, C84A, and C35A/C84A) hcTnC, as well as truncated N- and C-Domain hcTnC. ICP-OES was then used to accurately determine the relative Ca(II) and Cd(II) binding stoichiometry for each construct. Finally, CD studies were performed to determine the metal contribution for eliciting structural changes. Most importantly, the culmination of this data can provide information on the location of the third Cd(II) ion binding to N-Domain hcTnC. Ca(II) titrations into the full-length mutants revealed that the first two Ca(II) ions bind with similar thermodynamic parameters in all mutant constructs, but the third binding event has altered enthalpies and entropies, despite equal Gibbs free energy values. Cd(II) titrations into the full-length proteins revealed that the there are two exothermic heat events, with the first heat event binding much tighter than the second event and a total of approximately three Cd(II) ions binding. These Cd(II) titrations into mutant hcTnC also revealed that the cysteine residues do not play a key role in Cd(II) coordination, as previously suggested in the literature. Cd(II) titrations into truncated N-Domain hcTnC reveal one Cd(II) binding event, while Cd(II) titrations into truncated C-Domain HcTnC shows two Cd(II) binding events with different thermodynamic profiles, suggesting that Cd(II) does not bind in a positive cooperative mechanism. MD simulations of Cd(II) binding to truncated N-Domain hcTnC shows that Cd(II) is able to bind to Loop II of hcTnC, which is responsible for modulating function. In summary, our current Cd(II) binding model suggests that Cd(II) is binding to Loops III & IV of the C-Domain, as well as Loop II in the N-Domain. Taken together, the toxic effects of Cd(II) disruption can be pinpointed to its isostructural, but not isofunctional, role in metal mimicry.