Duality in disease: How two amino acid substitutions at actin residue 312 result in opposing forms of cardiomyopathy

Figures

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  Figure 1. Actin’s structure and substitutions at residue R312.A, the structure of monomeric G-actin, with its four subdomains (SD) colored (PDB 2BTF). B, the location of residue 312 (colored magenta) and the stabilizing interactions it forms. C, the structure of a 5 protomer polymeric actin filament, with a single protomer highlighted. D, the location of residue 312 (colored magenta) in relation to the neighboring F-actin protomers. E, the tropomyosin (Tm) binding conformations overlayed on an F-actin protomer with residue 312 (colored magenta) highlighted. Tm binds to actin filaments in the blocked (colored red, PDB 7UTL) state in the absence of calcium. In the presence of calcium, troponin pulls Tm into the closed (colored blue, PDB 7UTI) state. Upon myosin binding to F-actin, Tm is pushed into the open (colored green, PDB 8EFI) state.

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  Figure 2. In vitro results.A, an SDS-PAGE for the R312C variant following a co-sedimentation assay. Actin bands are on top, and tropomyosin bands are on the bottom. Tropomyosin bands increase in intensity along with tropomyosin concentration. B, co-sedimentation assay curves, fit using a specific binding with Hill slope in Prism GraphPad, of 7 μM ACTC proteins in the presence of Tm ranging from 0.1 to 2 μM Tm. N = 3 where N is a single co-sedimentation curve. Error bars represent SD. Three independent protein purifications were studied, with each being utilized for a single replicate. The Kd are listed in Table 1. C, kymographs of Alexa488-labeled tropomyosin binding along the length of an actin filament. A line was drawn along a filament and intensity measured over 60 s, increasing as Tm binds along the filament. Top: kymograph with WTRec; middle: R312C; bottom: R312H. For each kymograph, arrows represent the approximate start of intensity measurements following tropomyosin binding and elongation. Vertical lines represent the approximate start of peak intensity measurements. D, an average of all fluorescence increases over time, normalized from 1 to 100 percent. The time when fluorescence…

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  Figure 3. Structural properties from F-actin simulations. Chain C was isolated from each 5 protomer filament and analyzed. A, average root mean square deviation (RMSD) plot (N = 3 where N is an individual 200 ns replicate) displaying the average magnitude of changes in each structure over time, relative to the starting structure. WT (WT, colored black) plateaus around 50 ns, while R312C (red) and R312H (blue) diverge from WT and reach a plateau around 120 ns. B, the total number of protein-protein hydrogen bonds within the protomer over time (N = 3). R312C (red) displayed fewer bonds relative to WT (black) and R312H (blue), indicative of increased residue conformational freedom within the structure. The mean number of hydrogen bonds (±SD) for WT is 275 ± 7, R312C is 268 ± 8, and R312H is 277 ± 8. Differences in the average number of hydrogen bonds were found to be statistically significant (p < 0.0001) for all comparisons (WT versus R312C, WT versus R312H, and R312C versus R312H) following Kruskal–Wallis with Dunn’s Multiple Comparisons Tests. C, for a direct comparison of individual residue flexibilities, differences in average root mean square fluctuation (RMSF) are plotted (N = 3). RMSF…

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  Figure 4. Predicted structural changes from MD simulations. The most common structures of chain C from F-actin were isolated and analyzed. A, a cartoon representation of the WT protomer, showing its relative stability across the four subdomains. B, a cartoon representation of the R312C protomer, showing a forward swing of the Tm-bumper (residues 222–230) on the outside of SDs three-fourths as well as a forward shift of the D-loop in SD2. C, a cartoon representation of the R312H protomer, showing a forward swing of the Tm-bumper, with less magnitude than in R312C, as well as a backwards shift of the D-loop in SD2. D, a structural superposition of the five most common WT conformations (colored gray) to the starting structure (colored wheat), with the Tm-bumper and D-loop shown. The Tm-bumper is shifted slightly forward relative to the starting structure, but all five clusters assumed a similar conformation. The D-loop, with residues G46 (black), M47 (magenta), and K50 (green) colored for reference, exhibits some flexibility by moving in all directions. E, a structural superposition of the five most common R312C conformations (colored red) to the starting structure (colored wheat). The…

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  Figure 5. Actin interaction energies. Interaction energies were calculated between actin filaments conformations and docked ABPs myosin, troponin, and tropomyosin. For all plots, N = 10 where N is an actin structural cluster. All energy values are plotted as individual points, with bar graphs representing the average and error bars showing SD. A, myosin (colored cyan) in the open state was isolated from PDB 8EFI and docked to a 5 protomer filament through successive energy minimizations. Interaction energies were calculated between myosin and the two interacting chains (colored dark gray), the interacting SD3 in one protomer comprising residues 326 to 351 (colored red), and the interacting D-loop from one protomer (residues 38–53). B, average myosin:actin interaction energies were plotted for WT (gray), R312C (red), and R312H (blue). Average energy values are listed in Table S4. C, the troponin complex (TnI colored cyan, TnC colored dark blue, TnT colored purple) in the blocked state was isolated from PDB 7UTL was docked to an 8 protomer filament through successive energy minimizations. Interaction energies were calculated between the entire troponin complex and the 4 interacting actin…