Structural and functional consequences of cardiac troponin C L57Q and I61Q Ca2+-desensitizing variants

Abstract

Two cTnC variants, L57Q and I61Q, both of which are located on helix C within the N domain of cTnC, were originally reported in the skeletal muscle system [Tikunova and Davis (2004) J Biol Chem 279, 35341-35352], as the analogous L58Q and I62Q sTnC, and demonstrated a decreased Ca2+ binding affinity. Here, we provide detailed characterization of structure-function relationships for these two cTnC variants, to determine if they behave differently in the cardiac system and as a framework for determining similarities and differences with other cTnC mutations that have been associated with DCM. We have used an integrative approach to study the structure and function of these cTnC variants both in solution and in silico, to understand how the L57Q and I61Q mutations influence Ca2+ binding at site II, the subsequent effects on the interaction with cTnI, and the structural changes which are associated with these changes. Steady-state and stopped flow fluorescence spectroscopy confirmed that a decrease in Ca2+ affinity for recombinant cTnC and cTn complexes containing the L57Q or I61Q variants. The L57Q variant was intermediate between WT and I61Q cTnC and also did not significantly alter cTnC-cTnI interaction in the absence of Ca2+, but did decrease the interaction in the presence of Ca2+. In contrast, I61Q decreased the cTnC-cTnI interaction in both the absence and presence of Ca2+. This difference in the absence of Ca2+ suggests a greater structural change in cNTnC may occur with the I61Q mutation than the L57Q mutation. MD simulations revealed that the decreased Ca2+ binding induced by I61Q may result from destabilization of the Ca2+ binding site through interruption of intra-molecular interactions when residue 61 forms new hydrogen bonds with G70 on the Ca2+ binding loop. The experimentally observed interruption of the cTnC-cTnI interaction caused by L57Q or I61Q is due to the disruption of key hydrophobic interactions between helices B and C in cNTnC. This study provides a molecular basis of how single mutations in the C helix of cTnC can reduce Ca2+ binding affinity and cTnC-cTnI interaction, which may provide useful insights for a better understanding of cardiomyopathies and future gene-based therapies.

Computational Studies of the Effect of the S23D/S24D Troponin I Mutation on Cardiac Troponin Structural Dynamics

“…The coordinating time for Ser-69 was reduced equally for both models (Fig. 3 C), which is in agreement with our previous observation (16).…”

“…Based on these NMR structures, Lindert et al (12) and Kekenes-Huskey et al (13) studied the dynamics of NcTnC, as well as Ca 2þ association with NcTnC, via both conventional and accelerated molecular dynamics (MD) simulations, and examined the exposure dynamics and kinetics of cTnC hydrophobic residues via microsecond MD simulations (14). Wang et al (15,16) applied experimental and computational approaches to study the interactions of cTnC variants with altered Ca 2þ -binding affinities with the switch peptide of cTnI. In 2003, Takeda et al (11) published the first crystal structure of the human cTn complex in the Ca 2þ -saturated form.…”

Effects of Cardiac Troponin I Mutation P83S on Contractile Properties and the Modulation by PKA-Mediated Phosphorylation

“…10,34,37–40 The labeling efficiency (also known as percentage of labeling) was obtained by comparing the IANBD fluorophore versus protein concentration ratio. 10,39 The protein’s concentration was determined using the Bio-Rad protein assay (based on the Bradford method), and the IANBD fluorophore concentration was computed using the maximal absorbance of labeled protein at a wavelength ~481 nm dividing by IANBD’s extinction coefficient (21 000 M −1 cm −1 ).…”

“…10,34,39,40 Solution applied for this measurement contains (in mM) the following: 150 KCl, 20 MOPS, 3 MgCl 2 , 2 EGTA, and 1 DTT (pH 7.0). Fluorescence signal was recorded at ~530 nm during the titration of (1) Ca 2+ into 2 mL of $\mathrm{normalc}\mathrm{normalT}\mathrm{normaln}{\mathrm{C}}_{\mathrm{normalI}\mathrm{normalA}\mathrm{normalN}\mathrm{normalB}\mathrm{normalD}}^{\mathrm{normalC}35\mathrm{normalS}}$ (0.6 µM), or (2) cTnI variants (WT, S23D/S24D, P83S or P83S/S23D/S24D) into 2 mL of $\mathrm{normalc}\mathrm{normalT}\mathrm{normaln}{\mathrm{C}}_{\mathrm{normalI}\mathrm{normalA}\mathrm{normalN}\mathrm{normalB}\mathrm{normalD}}^{\mathrm{normalC}35\mathrm{normalS}}$ (0.6 µM) in the presence of Ca 2+ (100 µM).…”

Mass Spectrometric Immunoassays in Characterization of Clinically Significant Proteoforms

“…Some of the widely utilized biomarkers of disease, such as cystatin C [67,68], prostate-specific antigen [69] or cardiac troponin I [70,71], are known to exist as several forms in vivo . Other proteins, such as hemoglobin, serum amyloid A and numerous membrane proteins and enzymes, have genetic polymorphisms that induce expression of variable proteoforms, each unique for an individual.…”