The effect of magnesium on calcium binding to cardiac troponin C related hypertrophic cardiomyopathy mutants

Abstract

Cardiac troponin C (cTnC) is the calcium (Ca2+) sensing component of the troponin complex. Binding of Ca2+ to cTnC triggers a cascade of myofilament conformational changes that culminate in force production. Mutations in cTnC linked to hypertrophic myocardial myopathy (HCM) induce a greater degree and duration of Ca2+ binding, which may underly the hypertrophic phenotype. Recent evidence from our laboratories demonstrated novel modifications of cTnC Ca2+ binding by cellular magnesium (Mg2+) that we hypothesize may be of significance in promoting HCM.Regulation of contraction has long been thought to occur exclusively through Ca2+ binding to site II of cTnC. However, abundant cellular Mg2+ is a potential competitor for binding to the same sites; work by several groups also suggests this is possible. We have used isothermal titration calorimetry (ITC) to explore the thermodynamic properties associated with the interaction between Ca2+/Mg2+ and site II of cTnC; these experiments demonstrated that physiological concentrations of Mg2+ may compete with Ca2+ to bind site II of cTnC.In experiments reported here, we studied a series of mutations in cTnC thought to be causal in HCM. Three mutants (A8V, L29Q, and A31S) slightly elevated the affinity for both Ca2+ and Mg2+, whereas other mutants (L48Q, Q50R, and C84Y), that are closer to the C-terminal domain and surrounding the EF hand binding motif of site II had a more significant effect on affinity and the thermodynamics of the binding interaction.

Umbrella Sampling Simulations Measure Switch Peptide Binding and Hydrophobic Patch Opening Free Energies in Cardiac Troponin

“…Computational methods can facilitate the study of these processes . Previous efforts have been able to utilize molecular dynamics (MD) and free energy methods to study Ca 2+ binding to cTnC site II. − MD methods have also been able to investigate disruptions in dynamics upon the introduction of mutations that alter site II’s Ca 2+ sensitivity or mutations that have been linked with disease. − Enhanced sampling methods, umbrella sampling (US), and long timescale simulations have been utilized to study the energetic pathway for the transition between Ca 2+ -unbound and Ca 2+ -bound forms. − Simulations have also incorporated cTnI into the system to elucidate how other subunits in the cTn complex can affect the dynamics of cTnC. − Computer-aided drug discovery has also proven effective in helping identify potential therapeutics for heart disease by targeting the cTn complex. − …”

Computational Exploration and Characterization of Potential Calcium Sensitizing Mutations in Cardiac Troponin C

“…25 Additionally, we have used TI to accurately obtain the relative binding affinity for wild-type cNTnC and mutant D67A/D73A for calcium and magnesium ions, 42 as well as the relative binding affinity of Mg 2+ for several calcium sensitizing mutations such as L48Q and hypertrophic cardiomyopathy-associated mutants Q50R and C84Y. 43 It is important to note that increased calcium sensitization of the myofilament and the cardiac troponin T subunit have been linked to cardiac arrhythmias. 64−67 In cases of increased sensitization, the relaxation of cardiac muscle was impaired.…”

“…We successfully predicted the correct calcium binding affinity trends of those mutations compared to the wild-type. Much work has also been done to study the dynamics and energetics of the hydrophobic patch opening and the potential effect of mutations in this region. − Additionally, the dynamics and effects of mutations in other parts of the cTn complex, particularly cTnI, have been explored. − Finally, thermodynamic integration has been previously applied to study calcium and magnesium binding to site II. , In this work, we rationally explore several presumed Ca 2+ sensitizing mutations based on protein stability point mutation predictions and characterize their effects with ASMD and TI. We hope that this publication will be able to serve as a guide for potential future protein design studies.…”

“…34−41 Finally, thermodynamic integration has been previously applied to study calcium and magnesium binding to site II. 42,43 In this work, we rationally explore several presumed Ca 2+ sensitizing mutations based on protein stability point mutation predictions and characterize their effects with ASMD and TI. We hope that this publication will be able to serve as a guide for potential future protein design studies.…”

Umbrella Sampling Simulations Simultaneously Measure Switch Peptide Binding and Hydrophobic Patch Opening Free Energies in Cardiac Troponin

“…6 Previous efforts have been able to utilize molecular dynamics (MD) and free energy methods to study Ca 2+ -binding to cTnC site II. [7][8][9][10][11] MD methods have also been able to investigate disruptions in dynamics upon the introduction of mutations that alter site II's Ca 2+ -sensitivity or mutations that have been linked with disease. [12][13][14] Enhanced sampling methods, umbrella sampling, and long timescale simulations have been utilized to study the energetic pathway for the transition between Ca 2+ -unbound and Ca 2+ -bound forms.…”