X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles

Abstract

In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca²⁺-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 Å and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca²⁺-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 Å from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.

Keywords: Ca2+-regulation; Thin filament; X-ray fiber diffraction; skeletal muscle.

Figure 1.

X-ray diffraction patterns from live frog skeletal muscles at non-filament overlap length. (A) A comparison of diffraction patterns in the low- to medium-angle region between the relaxed and the activated states, and (B) a comparison of those in the medium- to high-angle region between them. The meridional axis (M) is coincided. E is the equatorial axis. The letter T with an index of the 384 Å repeat denotes the troponin-associated meridional reflections and the letter A with the axial spacing in Ångstrom unit, the representative thin filament-based layer lines.

Figure 2.

The measured intensity distributions of the thin filament-based layer lines in the relaxed state (blue) and upon activation (green). The layer lines (A1 to A26) are indexed to the basic repeat of ∼360 Å and the number in the parenthesis denotes the axial spacing.

Figure 3.

Searching parameters used in the model calculation. (A) Parameters for the optimal positioning of tropomyosin (white) around the fiber axis which is denoted by a star mark. r and θ are the radial distance and the azimuthal angle of tropomyosin around the fiber axis, respectively. The zero positions for the parameters are defined in the text. The positive direction of r is away from the fiber axis and the clockwise rotation is positive for θ. (B) Parameters used for the optimal positioning of the troponin core domain (left). r and θ are the radial distance and the azimuthal angle of the troponin core domain around the fiber axis, respectively. The parameters α, β and γ are the rotation angle around the x, y and z Cartesian coordinates, respectively. The N-terminus of the troponin core domain is located so that it connects with the C-terminus of TNT1 (right). The loci of these termini are depicted by red circles. The variable range of each parameter is given in Table1. F-actin is shown by blue balls, in which four subdomains of an actin molecule are colored by red (subdomain 1), orange (subdomain 2), magenda (subdomain 3) and pink (subdomain 4). Tropomyosin is shown by the two strands of white balls and the troponin three subunits are shown by light blue (TNC), green (TNI) and yellow balls (TNT).

Figure 4.

Modeling thin filaments with an unchanging Holmes F-actin. (A) A model with the V-shaped troponin core domain is oriented toward the pointed/M-line end of the actin filament. (B) A model with the troponin core domain is oriented toward the barbed/Z-band end of the actin filament. The upper side is toward the pointed/M-line end. The color assignment for the constituent molecules is the same as in Fig. 3. The calculated and observed layer line intensities are compared in the relaxed state (C) and in the activated state (D).

Figure 5.

Four subdomains of an actin molecule and division of 16 segments used in the modeling. (A) An actin molecule consists of four subdomains by Kabsch et al. (1990). Subdomains 1, 2, 3 and 4 are colored in red, orange, magenda and pink, respectively. (B) The division of the actin molecule into 16 segments. The sequential number of amino acid residues in the boundary between neighboring segments is written above the color bar and the corresponding segment number is written below the bar. (C) The surface display of the actin molecule devided into 16 segments. The number of the segments is indicated, and the color assignment for them is the same as in (B). The right panel is the view with the left one rotated by 180° around the vertical axis.

Figure 6.

The best-fit models of the thin filament structure in the relaxed and the activated states. (A) The model of the relaxed state and (B) the model of the activated state. The upper panel is a side view and the lower panel is a top view seen from the pointed/M-line end. Changes in the orientation and dispositions of the molecules upon activation are directed by arrows with the amount of changes. TM, tropomyosin and TN, troponin. The color assignment for the constituent molecules is the same as in Fig. 3.

Figure 7.

Comparison of the calculated intensities from the best-fit models and the observed intensities of the thin-filament-based layer lines. (A) The relaxed state and (B) the activated state. The layer line intensities of the 71 Å, 19.9 Å, 17,7 Å, 16.9 Å and 14.2 Å were mostly too weak to measure. They are denoted by small dotted curves. R_f for the eight layer lines in total was ∼11% in the relaxed state and ∼13% in the activated state.

Figure 8.

Dispositional changes of actin, tropomyosin and the troponin core domain in the best-fit models. (A) Structural change in actin. The central figure is viewed along the fiber axis (z axis), and the left and right ones are rotated by ±90° around the fiber axis. Subdomains 1, 2, 3 and 4 are colored as in Fig. 5. The top panel shows the changes from the Holmes actin in the best-fit model in the relaxed state. The bottom panel shows the changes in the transition of the relaxed state to the activated state. The direction of change of subdomains in actin is depicted by an arrow. (B) The positional change of tropomyosin (viewed from the pointed/M-line end). The left is in the relaxed state and the right is in the activated state. In each state, the radial distance of tropomyosin from the filament axis is indicated by yellow. In the activated state, the azimuthal angle change of tropomyosin around the filament axis is denoted by an arrow. (C) The dispositional and orientation changes of the troponin core domain. The left is in the relaxed state and the right is in the activated state. The top panel (“Top View”) is viewed from the pointed/M-line end, and the bottom panel (“Side View”) is viewed perpendicular to the filament axis (y axis). In the top panel, the radial distance of the troponin core domain from the filament axis is indicated by a red line. In the bottom panel, the change in the rotation angle around the y axis is denoted by a curved arrow. The color assignment for the constituent molecules is the same as in Fig. 3.

Figure 9.

Comparison of the calculated intensities from the best-fit models and the observed ones of the three troponin-associated meridional reflections with the repeat of 384 Å. The observed data were measured from the highly orientated sols of native thin filaments (Oda, unpublished data). (A) The relaxed state/in the absence of Ca²⁺ ions and (B) the activated state/in the presence of Ca²⁺ ions. The intensity normalization is made so that the total intensities of the three reflections in each state are indentical.