Clinically Divergent Mutation Effects on the Structure and Function of the Human Cardiac Tropomyosin Overlap

Abstract

The progression of genetically inherited cardiomyopathies from an altered protein structure to clinical presentation of disease is not well understood. One of the main roadblocks to mechanistic insight remains a lack of high-resolution structural information about multiprotein complexes within the cardiac sarcomere. One example is the tropomyosin (Tm) overlap region of the thin filament that is crucial for the function of the cardiac sarcomere. To address this central question, we devised coupled experimental and computational modalities to characterize the baseline function and structure of the Tm overlap, as well as the effects of mutations causing divergent patterns of ventricular remodeling on both structure and function. Because the Tm overlap contributes to the cooperativity of myofilament activation, we hypothesized that mutations that enhance the interactions between overlap proteins result in more cooperativity, and conversely, those that weaken interaction between these elements lower cooperativity. Our results suggest that the Tm overlap region is affected differentially by dilated cardiomyopathy-associated Tm D230N and hypertrophic cardiomyopathy-associated human cardiac troponin T (cTnT) R92L. The Tm D230N mutation compacts the Tm overlap region, increasing the cooperativity of the Tm filament, contributing to a dilated cardiomyopathy phenotype. The cTnT R92L mutation causes weakened interactions closer to the N-terminal end of the overlap, resulting in decreased cooperativity. These studies demonstrate that mutations with differential phenotypes exert opposite effects on the Tm-Tn overlap, and that these effects can be directly correlated to a molecular level understanding of the structure and dynamics of the component proteins.

Figure 1

Atomistic model of the cardiac thin filament. cTnI is colored blue, cTnC red, cTnT yellow, the C-terminal Tm green, and the N-terminal Tm orange. The subset contains the baseline structure of the Tm overlap region. The model structure for the Tm at the overlap assumes a form with orthogonal termini.

Figure 2

Time-resolved fluorescence from the cTnT 100C IAEDANS–Tm 271C DDPM FRET pair. (A) Fluorescence decay of the donor only sample (black) compared to the sample with the donor and acceptor (red). The increased rate of decay is indicative of FRET. (B) Residuals for the data shown in panel A when fit with a single discrete population undergoing FRET. (C) Residuals for the data shown in panel A when fit with two discrete populations undergoing FRET. This residual fit does not contain the periodic variance from the data that is observed in panel B.

Figure 3

Locations of labeled sites. On cTnT, IAEDANS is attached to either site 100 or site 127 as a FRET donor. On Tm, site 13, 265, 271, or 279 is used to link the FRET acceptor DDPM.

Figure 4

Adding calcium to a thin filament increases the distance between Tm and cTnT in the region adjacent to the C-terminal portion of the overlap. The magnitude of this change is around 2 Å. This is small in comparison with the larger-scale calcium-induced movements in the Tn core. We observe an overall lack of calcium-induced movement at the overlap itself.

Figure 5

Effect of the Tm D230N mutation and the cTnT R92L mutation on the FRET distances at the overlap shown as arrows, with a black contour representing the overall changes at the overlap in the presence of calcium. Tm D230N (red arrows) decreases the distance between cTnT and Tm at the center of the overlap (between cTnT 100 and Tm 271). cTnT R92L (green arrows) increases the distance between cTnT and Tm at the center of the overlap region. Both mutations result in an opposing compensatory change in distance between cTnT 127 and Tm 279. The change in overlap distance along with the compensation is represented by the black contour on cTnT.

Figure 6

Distances ± SE for the wild type (WT), Tm D230N, and cTnT R92L containing thin filaments using cTnT site 100 as the FRET donor site. *p < 0.05; **p < 0.01.

Figure 7

Distances ± SE for the wild type (WT), Tm D230N, and cTnT R92L containing thin filaments using cTnT site 127 as the FRET donor site. *p < 0.05; **p < 0.01; ****p < 0.0001; +p < 0.05; + +p < 0.01; +++p < 0.001.

Figure 8

DSC results for fully reconstituted thin filaments of (A) wild-type, (B) Tm D230N, and (C) cTnT R92L variants. The fwhm of the actin–Tm binding interaction is represented by the sharp peak near 47 °C and represents the cooperativity of the interaction. This interaction is outlined in the wild-type data with a dashed line. The wild-type complex peak (0.993 ± 0.01 °C) is wider than that of the peak from the Tm D230N complex (0.883 ± 0.01 °C) but narrower than that of the cTnT R92L complex (1.38 ± 0.14 °C). The standard deviation resulted from two or three independent experiments.

Figure 9

(A) Average structures of molecular dynamics simulations for wild-type, D230N, and R92L complexes. The Tm’s are shown as the wild type, while the positioning of the cTnT of the mutants is shown with the Tm’s aligned with the wild-type structures. The C-terminal end of Tm is colored green, the N-terminal end of Tm orange, wild-type cTnT yellow, D230N blue, and R92L red. (B) Closer view of the N-terminus of the cTnT. The cTnT D89 and R92 side chains that create the salt bridge that is eliminated by the R92L mutation are shown in ball-and-stick representation. Cyan spheres are carbon atoms, white spheres hydrogen atoms, blue spheres nitrogen atoms, and red spheres oxygen atoms.

Figure 10

Predicted positions of the labeling sites using the all-atom model of the thin filament (top). Schematic of the relative orientations of the labeling sites as revealed from FRET (bottom). cTnT 100 is in the same relative position in both models. cTnT 127 appears roughly equidistant in the FRET model between the C-terminal sites of Tm, compared to the model in which it is more distant from site 279 than from site 265.