Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca2+-binding affinity in a C-domain mutant of cardiac troponin C

Abstract

The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30–40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+ than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.

Familial Dilated Cardiomyopathy Associated With a Novel Combination of Compound Heterozygous TNNC1 Variants

“…CMPs reconstituted with a mixture of 50% WT and 50% D132N variant (WT/D132N) displayed significantly reduced myofilament Ca 2+ sensitivity of force generation (pCa 50 = 5.32 ± 0.03), with a rightward shift of 0.11 pCa units compared with CMPs reconstituted with 100% WT cTnC (pCa 50 = 5.43 ± 0.02) ( Table 2 and Figures 5A,B). Consistent with previous reports that compared 100% D145E with 100% WT (Landstrom et al, 2008;Pinto et al, 2009;Veltri et al, 2017a), we found that reconstitution with a mixture of 50% WT and 50% D145E (WT/D145E) significantly increased myofilament Ca 2+ sensitivity of tension (pCa 50 = 5.58 ± 0.04), with a leftward shift of 0.15 pCa units (Table 2 and Figures 5A,B). Interestingly, we observed no significant difference in myofilament Ca 2+ sensitivity of tension upon reconstitution of CMPs with a mixture of 50% D132N and 50% D145E (D132N/D145E) ( Table 2 and Figures 5A,B).…”

“…It was previously shown that cTnC-depleted CMPs reconstituted with cTnC-D145E increased Ca 2+ sensitivity of steady-state isometric force generation (Landstrom et al, 2008;Pinto et al, 2011a;Veltri et al, 2017a). However, to our knowledge, the effects of cTnC-D132N or mixtures of these cTnCs have not been previously reported.…”

“…of Ca 2+ solutions ranging from pCa 8.0 to 4.0 at 21 • C. Normalized [Ca 2+ ]-force data for each CMP were fit using nonlinear least squares regression to a 2-parameter Hill equation to obtain parameter estimates for pCa 50 and n Hill , as described (Veltri et al, 2017a).…”

“…The tertiary structure of cTnC is dominated by the two globular halves of the molecule, the C-and N-domains, which are connected by a flexible linker (Takeda et al, 2003;Li and Hwang, 2015). The C-domain binds divalent cations (Ca 2+ and/or Mg 2+ ) at two EF-hand motifs -referred to as sites III and IV based on their location in the primary sequenceand has important structural and modulatory roles in the sarcomere (Holroyde et al, 1980;Marques et al, 2017;Veltri et al, 2017a). The N-domain contains an evolutionarily defunct site I and regulatory EF-hand site II that normally triggers contraction upon Ca 2+ binding during systole (Holroyde et al, 1980).…”

Dynamic Equilibrium of Cardiac Troponin C’s Hydrophobic Cleft and Its Modulation by Ca2+ Sensitizers and a Ca2+ Sensitivity Blunting Phosphomimic, cTnT(T204E)

“…This implies all sensitizers tested in this study work by shifting the dynamic equilibrium of cTnC towards the fully open conformation, which possesses higher Ca 2+ affinity than closed cTnC. 9, 20 Despite this common mechanism of action each sensitizer showed distinct behavior which will be discussed in depth below.…”