Characterizing the Binding of Toxic Heavy Metal Cadmium to Human Cardiac Troponin C: Insight into Wild-type Conformational Dynamics via Hydrogen/Deuterium Exchange Mass Spectrometry and Thiol Quantification of Truncated N-domain Constructs Using Ellman’s Reagent

Abstract

Divalent cadmium, Cd(II), is a toxic heavy metal ion that can disrupt biochemical pathways through its ability to mimic essential metals or bind adventitiously to various proteins. An emerging body of evidence suggests that the soft acid Cd(II), contrary to the predicted behavior postulated by the Hard-Soft Acid-Base (HSAB) theory, often shows a binding preference for oxygen-rich sites in proteins, many of which natively bind the hard acid calcium, Ca(II). Given the demonstrated link between Cd(II) exposure and cardiovascular disease, human cardiac troponin C (hcTnC)—a Ca(II) binding EF-hand protein essential for heart muscle contraction—has been chosen as a model to characterize such Cd(II) binding. This study expands the current understanding of Cd(II) interaction with this system by examining the potential role of sulfhydryl-containing cysteine residues at positions 35 and 84—located in EF-hand loop I and the linker region, respectively—in disulfide formation or Cd(II) coordination. To explore these interactions, native-PAGE analyses and thiol quantification with Ellman’s reagent (also referred to as DTNB) were conducted on truncated wild-type and cysteine-replacement mutant N-domain hcTnC constructs. Furthermore, this study observed the conformational dynamics of full-length wild-type hcTnC in the presence and absence of Ca(II) or Cd(II) via hydrogen/deuterium exchange mass spectrometry (HDX-MS) to gain insight into structural changes that occur upon metal binding. DTNB and native-PAGE results indicate that in the truncated N-domain, disulfide bond formation is negligible, and Cd(II) does not bind to cysteine residues C35 or C84 at stoichiometric ratios, though it subtly interacts with both thiols (~20%) at excess metal concentrations. These findings support a recent model of Cd(II) binding to hcTnC, suggesting that these cysteines do not play a primary role in Cd(II) coordination. Additionally, peptide-level HDX-MS revealed that both Ca(II) and Cd(II) elicit the canonical reduction in exchange at predicted binding sites, consistent with the current model which suggests Cd(II) binds to Loops III and IV in the C-domain and Loop II in the N-domain. Moreover, both metals also induced non-canonical, although not unprecedented, promotion of deuterium uptake in regions away from the binding site of the N-domain, suggesting an allosteric response, albeit with distinct differences noted between the essential and toxic metals. Taken together, these findings advance the ongoing investigation into Cd(II) binding to hcTnC, offering new insights into how this heavy metal may exert its toxicity and impact subsequent protein-protein interactions within the troponin complex.