Relaxed tarantula skeletal muscle has two ATP energy-saving mechanisms

Figures

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  Figure 1.. The tarantula skeletal muscle myosin IHM. Related to PDB accession no.3JBH. (A) IHM with the BH and the FH shown in green and blue, respectively, and S2 shown in gray. (B) Superimposed motor domains of the IHM PDB 3JBH FH and BH as compared with the crystal structure of scallop myosin PDB accession no. 1QVI (in tan) representing the “prepower stroke” bent conformation (2.6 Å resolution; Argopecten irradians; Gourinath et al., 2003) with the BH and FH lever arms angled to allow the formation of the IHM (see Fig. 7). (C) Tarantula skeletal muscle lattice model showing a segment of relaxed tarantula skeletal muscle thick filament formed by two sets of BH (green) and FH (blue) helices above the myosin tails (gray), surrounded by 12 thin filaments (a). In the upper images, only the lateral thin filaments are shown. In b, the helices of FHs are removed, and in c, both sets of BHs and FHs are removed, leaving only the S2s and tails. Scale bars, 14.5 nm.

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  Figure 2.. XRD patterns from tarantula skeletal muscle. (A) Relaxed muscles of an ex vivo leg (left) and an isolated live muscle (right), recorded at 22.5°C. Equatorial, meridional, and off-meridional intensity profiles (as shown in the corresponding colored boxes) are seen in blue (top), orange (left), and green (right), respectively. The axial span of the blue box in the equator is ±0.00755 nm−1; the radial span of the orange box in the meridian is ±0.0081 nm−1; and the green box radial range span is from 0.038 nm−1 to 0.072 nm−1. (B and C) XRD patterns of relaxed ex vivo leg muscles (B) and isolated muscles (C) recorded from 10°C to 40°C.

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  Figure 3.. Variation of the intensities of layer lines and meridional and equatorial reflections and their spacings in live relaxed tarantula skeletal muscle with temperatures (t) from 8.5°C to 40°C. (A) Normalized IMLL4. (B–D) Normalized I1,0 (B) and I1,1+2,0 (C) and the change of the I1,1+2,0/I1,0 ratio ΔI1,1+2,0/I1,0 (D). (E–H) Normalized intensities of the third (E) and sixth (F) meridional reflections (M3 and M6, respectively) and their relative spacing changes (G and H). The residual values of IMLL4 are shown in A with black (20%) and white (40%) arrows. The average value and its SEM is shown for each measurement at each temperature. Normalization was against values measured at 22.5°C. The results are given as mean ± SEM.

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  Figure 4.. Intensities of off-meridional layer lines from XRD patterns of relaxed tarantula skeletal muscle. Integration area shown in Fig. 2 A, green box. (A and B) Example intensity traces from leg muscles in one leg ex vivo (A) and isolated live muscle (B). (C) Myosin head radius calculated from MLL4 profiles for temperatures <10°C, 22.5°C, and >30°C. (D) Normalized off-meridional axial profiles of the first MLL (LL1) parallel to meridian (boxed area in B), showing a shift of the first peak only at low temperature. (E) Peak LL1 spacings of the MLL1 profiles shown in D for temperatures <10°C, 22.5°C, and >30°C. The results are given as mean ± SEM. ns, not significant; **, P < 0.01; ****, P < 0.0001; two-tailed unpaired Student’s t tests.

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  Figure 5.. Intensity of reflections along the meridian from XRD patterns of relaxed tarantula skeletal muscle. Integrated area seen in Fig. 2 A, orange box. (A and B) Ex vivo leg muscles (A) and isolated live muscle (B). (C and D) Radial intensity profile of the third myosin reflection M3 (C) and normalized (D) at <12.5°C, 22.5°C, and >33.5°C. (E) Radial width of the M3 reflection for <12.5°C and 22.5°C. The results are given as mean ± SEM. ns, not significant.

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  Figure 6.. Proposed thermosensing mechanism for thick filaments of relaxed tarantula skeletal muscle. (A) Normalized IMLL4, I1,0, and I1,1+2,0 (from Fig. 3, A–C). The residual values of IMLL4 are shown with black (20%) and white (40%) arrows. (B) Proposed thermosensing mechanism. (c) At the top range 20–27.5°C, the BHs (green) and FHs (blue) exhibit the bent structure required to form IHMs and helices. The FHs can sway away (light gray head, double arrow) and back, docking on their partner BH IHM positions. Lowering the temperature from this top range (b to a) progressively changes the heads to the straight structure (pink heads), incapable of making IHMs, and thus becoming disordered, which occurs in the FHs with slow (S) turnover rates, while the BHs with a very slow (VS) turnover rate remain ordered (c to b). We propose that progressively increasing temperature from the top range affects only a fraction of the FHs, which become semipermanently disordered midway to the thin filaments, while the BHs remain ordered (c to e; see text). The very slow (>1,800 s), slow (250–300 s), and fast (<30 s) ATP turnover rates measured in relaxed tarantula skeletal muscle thick filaments (Naber et al.,…

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  Figure 7.. Two ATP energy-saving mechanisms proposed in tarantula skeletal muscle. A cross-bridge cycle for tarantula skeletal muscle, based on previous work by Robert-Paganin et al. (2020), is shown and includes actin-detached steps (yellow box) and actin-attached steps (green box). (A) The energy-saving IHM (PDB accession no. 3JBH) allows quick incorporation of the detached, swaying FHs to the cycle (Padrón et al., 2020). The FHs, which are in the bent, prepower stroke structure (similar to PDB accession no. 1QVI, shown in tan in steps 1 and 2 and in Fig. 1 B) containing Mg.ADP.Pi (Zoghbi et al., 2004), can sway away and back by Brownian motion (Brito et al., 2011; step 1) and weakly attach to the switched-off thin filaments (step 2). When the thin filament is switched on by the tropomyosin/troponin Ca2+ switch (Craig and Lehman, 2001), the FHs attach strongly, going through the power stroke and producing force and displacement (step 3) transitioning, after Pi and then ADP release, to a nucleotide-free rigor/rigor-like structure similar to PDB accession no. 2OS8 (step 4). The binding of Mg.ATP to the rigor/rigor-like FHs induces fast dissociation of the heads from actin (step 5), followed…