The Interacting Head Motif Structure Does Not Explain the X-Ray Diffraction Patterns in Relaxed Vertebrate (Bony Fish) Skeletal Muscle and Insect (Lethocerus) Flight Muscle

Figures

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  Figure 1. (a) The interacting head motif (IHM) of the two myosin heads in a myosin molecule (pdb accession code 3DTP) shown as a stereo pair (wall-eyed stereo). One head in each pair is in shades of blue and the other in yellow. The motor domains are towards the top, and the lever arms with a long central α–helix (each with two light chains, yellow and red on the right head) are below the motor domains. The coiled-coil of part of the subfragment 2 (S2) of the myosin rod, where the two heads join together, is shown at the lower left. The actin binding site on the right head abuts the left head. The right hand head is called the blocked head and the other the free head. (b) Stereo image of the reconstruction from electron microscopy and single particle analysis of the myosin filaments in human heart muscle, with heads fitted to the density. The total length of filament shown here is around 43 nm. There are three crowns of heads with six heads in each crown. The head pair configuration is similar to that in (a), but the whole structure, for example of the heads nearest to the viewer, has been rotated 180° top to bottom roughly around a horizontal rotation axis in the plane of the page. Other…

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  Figure 2. (a) The low-angle X-ray diffraction pattern from bony fish muscle (fibre axis vertical) showing the horizontal layer-lines and vertical row-lines on which the observed peaks are situated. Almost all of what can be seen here comes from the myosin filaments in the muscle. (b) The “simple lattice” unit cell of the A-band lattice in bony fish muscle viewed down the fibre axis showing the single rotation (N) of the myosin filaments around their long axes and the 3-fold rotational symmetry of the head arrangement on one crown of heads. Actin filament positions (black circles) at the trigonal points are labelled A.

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  Figure 3. The fish muscle unit cell with the myosin filament head arrangements (dark yellow) from (a) Hudson et al. [10] and (b) as in human heart muscle (AL-Khayat et al. [25]). The myosin filament backbone structure is from Chew and Squire [30]. Actin filaments are shown in green.

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  Figure 4. The calculated R-factor from the human heart muscle myosin filament in the fish muscle unit cell (red trace) showing a large variation with rotation angle relative to the unit cell axis. The analogous R-factor for the fish thick filament structure determined by Hudson et al. [10] is in blue. As expected, both R-factors are periodic because of the symmetry in the lattice and they repeat after 60°. Also included is the R-factor of the hybrid human system if the myosin S2 is also included. Inclusion of S2 gave a slightly poorer fit, but both human models with the interacting heads motif give very poor R-factors compared to the structure from Hudson et al. [10]. The R-factor for the chimera model was slightly improved if the 2D temperature factor was included (see text).

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  Figure 5. (a) Simulated X-ray diffraction pattern using the observed corrected intensities from bony fish muscle (cf. Figure 2a). (b) Calculated diffraction pattern using the myosin filament structure (Figure 3a) from Hudson et al. [10] that is the best fit to (a); R-Factor 7.9%. (c) The calculated X-ray pattern if the human heart muscle myosin filaments with the interacting head motif (AL-Khayat et al. [26]) are put into the fish muscle unit cell instead of the structure from Hudson et al.; lowest R-Factor = 54%. Inclusion of myosin S2 in the calculation in (c) makes the fit slightly less good (see Figure 4). All patterns were scaled to have the same total intensity. Introduction of a small lateral disorder factor (2D temperature factor) slightly improved the R-factor in (c) (with S-2) to 32% with a root mean squared lateral displacement of the myosin filament from its ideal lattice point of 22 Å.

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  Figure 6. (a) The observed low-angle X-ray diffraction pattern from insect flight muscle (Lethocerus; fibre axis vertical) courtesy of R.J. Edwards and M.K. Reedy [7,8,9] and (b) the insect flight muscle unit cell viewed down the fibre axis, showing the unique orientation of the myosin filaments in the lattice and their 4-fold rotational symmetry on a single crown of heads. Myosin heads are represented as shaded or white ovals. Actin filaments, all halfway between adjacent myosin filaments, are labelled as A. Compare Figure 2b for vertebrate striated muscles. The rotation N in the lattice is ill-defined (see text).

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  Figure 7. Part of the 3D reconstruction of the insect flight muscle myosin filament according to Hu et al. [27], here showing slightly more than three crowns as a stereo (wall-eyed) surface view.

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  Figure 8. 3D structure of the insect flight muscle myosin filament according to Hu et al. [27] shown as stereo (wall-eyed) side views containing three crowns. (Top) Surface view of the density map, low pass Fourier filtered to 25-Å resolution, with fitted coordinates of atomic models of myosin heads in the interacting head motif arrangement, as determined in the present work, shown in cartoon representation. (Bottom) The atomic models without the density map. Coordinates colour-coded as follows: Free head heavy chain—cyan, essential light chain—magenta, regulatory light chain—straw: Blocked head heavy chain—green, essential light chain—orange, regulatory light—yellow.

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  Figure 9. The models in Figure 8 shown looking down the filament axis, with the same colour coding. (Top) Density plus fitted model; (Bottom) Fitted model only. The head pairs form a good fit with a closed structure, as in the interacting head motif.

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  Figure 10. Comparison of three crown levels of the Hu et al. [27] insect flight muscle myosin filament density map (a) and the model (b) from AL-Khayat et al. [11] based on fitting X-ray diffraction data.

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  Figure 11. Comparison of the observed low-angle X-ray diffraction pattern (a) from relaxed insect (Lethocerus) flight muscle after processing (upper half of Figure 6a) and the simulated diffraction patterns from various versions of the insect myosin filament in the unit cell: (b) The published model from AL-Khayat et al. [11], R-factor = 9.7%; (c) The density map from Hu et al. [27] (Figure 7) put into the insect unit cell; R-factor = 33%, (d) The fitted atomic model with the interacting heads motif (Figure 8 and Figure 9), R-factor = 27%. (b–d) used the R-factor given in Equation (3). Layer-line numbering is based on the approximate 1160-Å axial repeat in insect flight muscle. Layer-line 3 was not used for the R-factor calculations, because both actin and troponin contribute to that layer-line as well. All patterns were scaled to give the same total intensity in the pattern, apart from (b) which is modified from the results of AL-Khayat et al. [11] and scaled to look similar to the other patterns. Introduction of a lateral (2D) disorder factor slightly improved the R-factors in (c) and (d) to 21% and 19% with a root mean squared displacement of the myosin filament from its ideal lattice…

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  Figure 12. (a) One crown of the IHM (super-relaxed?) head configuration on the myosin filaments of insect flight muscle, looking M-wards and showing the interacting head motif (cf. Figure 9). (b) One crown of the resting insect flight muscle myosin filament (slightly refined from) reported by AL-Khayat al [11], at the rotation around its long axis that best fits with the structure from Hu et al. [27], with the S2 positions coincident and showing one head near the filament surface and the other projecting radially outwards from the filament backbone (shown here as an empty space). (c) The structures in (a,b) are superimposed to show the similar, overlapping, locations of one of each head pair and the changed positions of the other heads between the two structures. The white arrows indicate, for the free head swing model, the swing of the free heads around the S2 positions, which are shown as small yellow dots, between what we assume is the super-relaxed state and what we now think of as the “activated” relaxed state. In an alternative mechanism (not illustrated), the head switch mechanism, the outer blocked head in the IHM structure moves to project radially outwards and the inner free IHM…