Relaxed and active thin filament structures; a new structural basis for the regulatory mechanism

Fig. 1. Comparison of data from Ca2+-free and Ca2+-treated filaments. (A and B) Data from Ca2+-treated filaments, (C and D) data from Ca2+-free filaments. (A and C) The five columns represent different viewing angles of the filaments described by the Euler angles α = 0°, β = 90° throughout and then with the angles γ shown below each figure. Rows Ai and Ci are class averages, Aii and Cii are reprojections and Aiii and Ciii are surface views of the 3D reconstruction displayed at the corresponding angles. The cross-correlation coefficient between the class averages and the corresponding reprojections are given at the top of each column for the Ca2+-treated state (A) and the corresponding cross-correlations between the inverted class average and the reprojection are shown underneath in brackets. Similar cross-correlations are shown in C for the Ca2+-free state (C). (B and D) The surface rendered 3D maps of the Ca2+-treated filament (B) and the Ca2+-free filament (D). Two viewing angles of the reconstructions are shown with a 90° rotation about the filament axis between the two.

Fig. 2. Difference density analysis. (A–C) Data from Ca2+-treated filaments, (D–F) data from Ca2+-free filaments. (A and D) Wire mesh representation of the single particle based reconstructions of the thin filament in the two states. An atomic F-actin model is docked into the reconstruction and each subunit is colour coded. The barbed end (Z-band end) of the actin filament is at the bottom of the figure. (B and E) Difference density maps calculated by subtracting the docked F-actin model (grey) from the single particle reconstructions. This leaves the density attributable to the regulatory proteins troponin and tropomyosin (both orange). (C and F) Difference density maps calculated by subtracting docked F-actin (grey) and tropomyosin (orange) models from the single particle reconstructions leaving density attributable only to troponin (blue).

Fig. 3. Fitting the troponin density. (A–D) Data from Ca2+-treated filaments, (E–H) data from Ca2+-free filaments. (A and E) The density attributable to troponin is shown (blue) in the Ca2+-treated state (A) and Ca2+-free state (E). (B, C and F, G) Space filling (B and F) and ribbon diagrams (C and G) of models of the troponin complex based on the crystal structures of the core domains: 1YVO for the Ca2+-free state and 1YVTZ for the Ca2+-treated state (Vinogradova et al., 2005) together with the atomistic model of the whole complex (Manning et al., 2011) that returned the best fit docked into the electron density envelopes produced by the difference analysis. The three troponin components are displayed in different colours: TnC, blue; TnT green; TnI pink. (C and G) The location of the C-terminal region of TnI is highlighted by a star, TnT1 is indicated by a triangle and the ordered central helix of TnC is arrowed in C. (D and H) Maps of the Ca2+-treated (D) and Ca2+-free (H) filaments with all the components docked into the density (tropomyosin shown in orange).

Fig. 4. Visualisation of the tropomyosin domain movements and myosin binding. (A) Structures of the Ca2+-free state and (B) structures of the Ca2+-treated thin filament assembly. (A and B) show the central region of the Ca2+-free (A) and the Ca2+-treated (B) reconstructions over a length of 14 actin subunits, two strands of tropomyosin (orange) and two troponin complexes. The core domains of the COMPHI model are in magenta. Subunits a to g of one strand of actin are colour coded and the second strand is grey. In the Ca2+-free state (A) the position of tropomyosin is the same on every subunit and lies in the B or blocked position where it would inhibit myosin binding. The position of tropomyosin is different on different subunits of the actin filament in the Ca2+-treated state (B), where the average positions are illustrated for actin subunits d to g (closed state) and a to c (M-state). A & B (right inserts) show face on views of the actin subunits, with weak (blue) and strong (red) actin binding sites highlighted for the three distinct positions of tropomyosin. Subunits a, b and c show tropomyosin in the “M or Myosin state”, subunits d, e, f, g show tropomyosin in a position more closely…

Fig. 5. A novel mechanism of regulation. Schematic diagrams of the thin filament assembly for simplicity shown as a linear array (i.e. not helical). Protein components colour coded: (yellow), tropomyosin (blue) and troponin (TnC, speckled blue; TnI, red; TnT green). When the thin filament changes from the Ca2+ free state (a) to the Ca2+ bound state (b) the tropomyosin strands on actins g, a and b move further than those on actins c-f. Actins g, a and b may be exposed enough for some myosin heads to bind (dashed outline). Bound heads going over to strong states may then activate the whole filament (c). The body of troponin may move out of the way (dashed arrows) to permit binding of further heads. (a and b) also illustrate a potential mechanism whereby the distal arm of TnT1 acting on the tropomyosin overlap region may swing to expose the binding sites on actins g, a and b when TnC binds Ca 2+.