Myosin loop-4 is critical for optimal tropomyosin repositioning on actin during muscle activation and relaxation

Figures

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  Figure 1.. Cryo-EM images. (A and B) Actin-tropomyosin filaments were decorated with wild-type human cardiac myosin S1 (A) and with mutant 7G S1 (B). Unbound tropomyosin and myosin can be seen in the background. Scale bar, 50 nm.

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  Figure 2.. Cryo-EM reconstructions of S1-decorated actin-tropomyosin filaments. (A–C) Isosurface rendering of the reconstructions containing wild-type S1 (A; blue) and 7G-mutant S1 (B; gold); in C, A and B are superposed, showing tropomyosin positions are different in the two reconstructions. The pointed ends of the filaments in the reconstructions are facing up, with six S1 and actin subunits and corresponding tropomyosin coiled coils shown. In A, tropomyosin is marked by a horizontal open arrow, the bottom-right S1 and actin subunits are indicated by oblique and vertical open arrows, respectively. Myosin loop-4 is most easily noted extending from the centrally located S1 density toward tropomyosin (white arrows, also see Fig. 3), while in A projecting densities representing side chains of myosin residues 369 and 368 at the loop-4 tip are apparent. Loop-4 in the glycine-substituted 7G-S1 (B), lacking these side chains, has a smooth contour. (D and E) Atomic models of F-actin, S1, and tropomyosin filaments were fitted to the front-facing filament strand of reconstructions shown in A and B; here, the reconstruction isosurface was made transparent in order to examine the atomic models; note…

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  Figure 3.. Myosin loop-4–tropomyosin interaction. (A–F) Loop-4 structure in reconstructions of S1-decorated actin-tropomyosin filaments containing wild-type S1 (A–C) and 7G-mutant S1 (D–F). (A and D) Here, the reconstructions in Fig. 2 were tilted orthogonally with the filament pointed ends facing away from the viewer and transverse sections of the reconstructions then made at the level of loop-4. (B and E) Atomic models of F-actin, S1, and tropomyosin filaments fitted within the corresponding EM volumes; with C showing A and B superposed, and F the same for D and E. The vertical open arrows mark loop-4 and in A highlights projecting densities likely to represent side chains of wild-type myosin residues Arg 369 and Gln 368 approaching the tropomyosin coiled coil (right-angle arrow). The loop is discontinuous in the 7G-S1 (D) likely reflecting disorder in the region of the glycine substitution. Horizontal open arrow points to the neighboring actin surface. Scale bar, 5.0 Å.

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  Figure S1.. Image reconstruction procedure and reconstruction quality. (A) Flowchart summarizing steps taken to process cryo-EM images in order to generate a 3-D reconstruction of F-actin–tropomyosin decorated with human cardiac S1, in which loop-4 residues 366–372 were mutated to glycine. (B) Map-to-model fit of atomic models of filament components to the reconstruction, here focused on five centrally located actin protomers, an associated segment of tropomyosin, and one S1 motor domain. (C) Regions of the fitted reconstruction magnified to indicate the quality of the data showing selected chains within actin and myosin as well as the ADP bound to actin, rendered in ChimeraX. (D) Fourier shell correlation of the split reconstruction data sets, indicating 3.6 Å resolution at a value of 0.143.

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  Figure 4.. Tropomyosin movement from M-state positions during MD. To mimic the movement of tropomyosin following release of myosin from F-actin, MD simulations were initiated for atomic models of tropomyosin localized on F-actin as in reconstructions decorated with either WT-S1 or 7G-S1 (see Fig. 2, D and E), but here with no S1 present. The green reference line in the diagram shows the positions of tropomyosin at the beginning of MD (the 3–4 Å difference in two tropomyosin starting positions is not highlighted here). The blue and yellow bands show positions explored by tropomyosin cables during MD starting with those in respective wild-type and 7G-associated models. The orange line indicates the C-state position of tropomyosin (Yamada et al., 2020) approached by tropomyosin beginning from the wild-type position (blue band). Tropomyosin starting from the aberrant M-state induced by 7G-S1 rarely contacts the C-state position; on average it remains in place and in fact often moves towards the extreme inner domain of F-actin (yellow band), viz. in a path opposite to that normally expected.