Formation of an Amyloid-like Structure During In Vitro Interaction of Titin and Myosin-Binding Protein C

Figures

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  Figure 1. Schematic structure of a muscle sarcomere with thick and thin filaments. MyBP-C is localized in the C-zone and is highlighted in green; titin is shown in yellow.

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  Figure 2. Localization of MyBP-C and titin molecules in the sarcomere. MyBP-C lies transversely to the axis of the thick myosin filament, forming 9 regularly located stripes with an interval of about 43 nm between the super-repeats of the C-zone of titin, starting from C2–C3 (the second and third super-repeats of the C-zone of titin) to C10–C11 (the 10th and 11th super-repeats of the C-zone of titin). However, between the last super-repeat of the D-zone and the first super-repeat of the C-zone of titin and between the super-repeats C1–C2, MyBP-C stripes are absent. According to the literature, three domains of MyBP-C (FnIII2, Ig7, and FnIII3) are associated with three domains of titin (FnIII10, FnIII11, and Ig1) at 11 sites of the titin molecule [4,7].

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  Figure 3. Electron microscopy of titin (A), MyBP-C (B), and titin/MyBP-C complexes (C) formed in a solution of 175 mM KCl and 10 mM imidazole, pH 7.0. Insets 1 and 2, enlarged views of informative regions. Inset 1, titin filaments presumably decorated with MyBP-C molecules. Inset 2, a single filament of presumably titin (marked with black arrows) emerging from the aggregate. Decoration elements, presumably MyBP-C molecules, on part of the filament (marked with black checkmarks). Quantitative analysis of electron microscopy images (Supplementary Figure S3, Supplementary File S1) showed that the average diameters of aggregates in regions I, II, and III were 36.9 ± 7.5 nm, 29.9 ± 5.5 nm, and 28.4 ± 5.8 nm, respectively, indicating a compact organization of the structures and a specific morphology of the detected aggregates. Negative staining with 2% aqueous uranyl acetate.

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  Figure 4. Atomic force microscopy of titin/MyBP-C complexes (A–C) formed in 175 mM KCl and 10 mM imidazole, pH 7.0. Dialysis was performed for 24 h at 4 °C. The ratio of MyBP-C to titin was 1:1. The final protein concentration was 0.12 mg/mL. Time of formation of aggregates and complexes at 4 °C, 24 h. Statistical analysis of the images (height, roughness, asymmetry, and tilt angles) is presented in the Supplementary Materials (Supplementary Figure S5, Supplementary File S4). The obtained values indicate the presence of localized relief differences with an overall uniform surface, which corresponds to amorphous or compact aggregation.

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  Figure 5. Confirmation of the amyloid-like structure in titin/MyBP-C complexes. (A) X-ray diffraction of titin/MyBP-C complexes after 24 h of co-incubation in 175 mM KCl and 10 mM imidazole, pH 7.0. Two diffuse ring reflections at 4.7 and 10 Å corresponding to the cross-β structure were detected. (B) Fluorescence intensity of ThT in the presence of titin (blue), MyBP-C (brown), and titin/MyBP-C complexes (gray) formed during 24 h in a solution containing 175 mM KCl and 10 mM imidazole, pH 7.0. The fluorescence intensity of ThT (yellow) was used as a control. (C) FTIR spectra of titin, MyBP-C, and titin/MyBP-C complexes at 20 °C. Protein concentration, 10–15 mg/mL. Green, non-aggregated skeletal muscle titin; blue, non-aggregated skeletal muscle MyBP-C; gray, titin/MyBP-C complexes formed in 175 mM KCl and 10 mM imidazole, pH 7.0.

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  Figure 6. Predicted amyloidogenicity of titin and skeletal/cardiac MyBP-C. (A) Full-length titin (34,350 amino acid residues) composed of 152 immunoglobulin-like (Ig) and 132 fibronectin type III (FnIII) domains. (B) Skeletal MyBP-C (1141 residues) consisting of seven Ig-like and three FnIII-like domains. (C) Cardiac MyBP-C (1274 residues) with the same domain organization. Colored bars represent predicted amyloidogenic regions within individual domains, as determined by FoldAmyloid and AGGRESCAN algorithms. Highlighted domains correspond to known interaction sites between titin and MyBP-C.