In depth, thermodynamic analysis of Ca2+ binding to human cardiac troponin C: Extracting buffer-independent binding parameters

Johnson, Rachel A.; Fulcher, Lindsay Michelle; Vang, Katie; Palmer, Caitlin D.; Grossoehme, Nicholas E.; Spuches, Anne M.

doi:10.1016/j.bbapap.2019.01.004

Cited by 8 publications

(7 citation statements)

References 31 publications

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“…As seen in previous reports, we found the binding of Ca 2+ to N-cTnC to be driven by entropy and unfavorable enthalpy ( Table S2 ) ( 64 , 65 ). The favorable ΔS may be due in part to the hydration enthalpy of Ca 2+ , which is thought to be on the order of ∼375 kcal ∗ mol −1 and slightly lower than that of Mg 2+ (∼460 kcal ∗ mol −1 ) ( 67 ).…”

Section: Discussionsupporting

confidence: 86%

“…Binding of Ca 2+ /Mg 2+ to site II is characterized by an endothermic interaction as indicated by our titrations on the N-terminal domain in this and previous publications ( 63 , 64 ). From this and the ITC work on full-length cTnC by others ( 65 ), we can deduce that the exothermic heat changes seen in Figure 6 result from interactions with site III/IV. The data ( Fig.…”

Section: Resultssupporting

confidence: 61%

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Binding of calcium and magnesium to human cardiac troponin C

Rayani

Seffernick

et al. 2021

Journal of Biological Chemistry

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Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca 2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca 2+ -sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca 2+ and Mg 2+ and a regulatory site (II) that has been thought to bind only Ca 2+ . Binding of Ca 2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca 2+ /Mg 2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca 2+ /Mg 2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg 2+ caused a 1.4-fold lower affinity for Ca 2+ . These experiments strongly suggest that cytosolic-free Mg 2+ occupies a significant population of the available site II. Interaction of Mg 2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg 2+ , such as secondary hyperparathyroidism or ischemia, respectively.

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Section: Discussionsupporting

confidence: 86%

Section: Resultssupporting

confidence: 61%

Binding of calcium and magnesium to human cardiac troponin C

Rayani

Seffernick

et al. 2021

Journal of Biological Chemistry

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show abstract

“…As seen in previous reports, we found the binding of Ca 2+ to N-cTnC to be driven by entropy and unfavorable enthalpy (Table S2) (Stevens, Rayani et al 2017, Johnson, Fulcher et al 2019). The favorable ΔS may be due in part to the hydration enthalpy of Ca 2+ which is thought to be on the order of ~375 kcal*mol -1 and slightly lower than that of Mg 2+ (~460 kcal*mol -1 ) (Smith DW, 1977).…”

Section: Discussionsupporting

confidence: 85%

“…Binding of Ca 2+ /Mg 2+ to site II is characterized by an endothermic interaction as indicated by our titrations on the N-terminal domain in this and previous publications (Stevens, Rayani et al 2016, Stevens, Rayani et al 2017. From this, and ITC work on full-length cTnC by others (Johnson, Fulcher et al 2019), we can deduce that the exothermic interactions seen above (Figure 5) result from interactions with site III/IV. The data (Figure 5 and Table S3) show that Mg 2+ binds to apo-state fulllength cTnC at two distinct sets of sites.…”

Section: Ca 2+ Binding To Apo-state Full Length Ctncsupporting

confidence: 83%

Binding of Calcium and Magnesium to Cardiac Troponin C

Rayani

Seffernick

et al. 2020

Preprint

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Cardiac troponin C (cTnC) is the Ca 2+ -sensing component of the thin filament. It contains structural sites (III/IV) which bind both Ca 2+ and Mg 2+ , and a regulatory site (II) that has been thought to bind only Ca 2+ . The latter binding initiates a series of conformational changes that culminate in force production.We have quantified the interaction between site II and Ca 2+ /Mg 2+ through Isothermal Titration Calorimetry and Thermodynamic Integration simulations. Direct and competitive binding titrations using wild type and a double mutant that significantly reduces binding to site II demonstrated that physiologically relevant concentrations of both Ca 2+ /Mg 2+ interact with the same locus. Cytosolic free Mg 2+ (~1 mM) could occupy a significant population of available site II, as this concentration of Mg 2+ decreased the affinity for Ca 2+ 1.4-fold.Interaction of Mg 2+ with site II of cTnC likely has important functional consequences for the heart at baseline and in diseased states which decrease or increase availability of Mg 2+ such as secondary hyperparathyroidism or ischemia, respectively.

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“…The experimental change in enthalpy (Δ H ITC ) and the association constant ( K a, ITC ) were corrected for buffer competition in a post hoc analysis to yield buffer-independent thermodynamic parameters for Zn–Peptide binding. The post hoc analysis described below follows protocols previously applied to many metallopeptide and metalloprotein systems. ,− ,− …”

Section: Methodsmentioning

confidence: 99%

Multiple Modes of Zinc Binding to Histatin 5 Revealed by Buffer-Independent Thermodynamics

et al. 2023

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Histatin 5 (Hist5) is an antimicrobial peptide found in human saliva as part of the innate immune system. Hist5 can bind several metal ions in vitro, and Zn 2+ has been shown to function as an inhibitory switch to regulate the peptide's biological activity against the opportunistic fungal pathogen Candida albicans in cell culture. Here, we studied Zn 2+ binding to Hist5 at four temperatures from 15 to 37 °C using isothermal titration calorimetry to obtain thermodynamic parameters that were corrected for competing buffer effects. Hist5 bound Zn 2+ with a buffer-dependent association constant of ∼10 5 M −1 and a bufferindependent association constant of ∼6 × 10 6 M −1 at pH 7.4 and at all temperatures tested. Zn 2+ binding was both enthalpically and entropically favorable, with larger entropic contributions at 15 °C and larger enthalpic contributions at 37 °C. Additionally, the Zn:Hist5 binding stoichiometry increased from 1:1 to 2:1 as temperature increased. The enthalpy−entropy compensation and the variable stoichiometry lead us to propose a model in which the Zn−Hist5 complex exists in an equilibrium between two distinct binding modes with different Zn:Hist5 stoichiometries. The in-depth thermodynamic analysis presented herein may help illuminate the biophysical basis for Zn-dependent changes in the antifungal activity of Hist5.

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In depth, thermodynamic analysis of Ca2+ binding to human cardiac troponin C: Extracting buffer-independent binding parameters

Cited by 8 publications

References 31 publications

Binding of calcium and magnesium to human cardiac troponin C

Binding of calcium and magnesium to human cardiac troponin C

Binding of Calcium and Magnesium to Cardiac Troponin C

Multiple Modes of Zinc Binding to Histatin 5 Revealed by Buffer-Independent Thermodynamics

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