Tropomyosin position on F-actin revealed by EM reconstruction and computational chemistry

Abstract

Electron microscopy and fiber diffraction studies of reconstituted F-actin-tropomyosin filaments reveal the azimuthal position of end-to-end linked tropomyosin molecules on the surface of actin. However, the longitudinal z-position of tropomyosin along F-actin is still uncertain. Without this information, atomic models of F-actin-tropomyosin filaments, free of constraints imposed by troponin or other actin-binding proteins, cannot be formulated, and thus optimal interfacial contacts between actin and tropomyosin remain unknown. Here, a computational search assessing electrostatic interactions for multiple azimuthal locations, z-positions, and pseudo-rotations of tropomyosin on F-actin was performed. The information gleaned was used to localize tropomyosin on F-actin, yielding an atomic model characterized by protein-protein contacts that primarily involve clusters of basic amino acids on actin subdomains 1 and 3 juxtaposed against acidic residues on the successive quasi-repeating units of tropomyosin. A virtually identical model generated by docking F-actin and tropomyosin atomic structures into electron microscopy reconstructions of F-actin-tropomyosin validated the above solution. Here, the z-position of tropomyosin alongside F-actin was defined by matching the seven broad and narrow motifs that typify tropomyosin's twisting superhelical coiled-coil to the wide and tapering tropomyosin densities seen in surface views of F-actin-tropomyosin reconstructions. The functional implications of the F-actin-tropomyosin models determined in this work are discussed.

Figure 1

Electron micrographs of negatively stained F-actin-tropomyosin. Note the obliquely oriented tropomyosin strands (arrows). Filaments are shown with their pointed ends facing up; polarity was determined with alignment tools as previously described (27). Scale bar: 50 nm.

Figure 2

Fitting the atomic model of F-actin into the F-actin-tropomyosin reconstruction. (a) F-actin-tropomyosin reconstruction. Actin subdomains are marked on two actin subunits. An arrow indicates the tropomyosin strand (and points toward the pointed end of the filament), and the open white arrows point to locations where the tropomyosin strand coils appear to cross over each other. (b) Tropomyosin difference density (magenta) superposed on the F-actin-tropomyosin reconstruction. (c) Tropomyosin model fitted in tropomyosin difference density. (d) Three actin subunits from the Oda atomic model of F-actin (22) fitted into the reconstruction in b, with the actin part of reconstruction now made transparent. (e) Atomic model of actin as in d, with just the superposed tropomyosin difference density made transparent. (f) Atomic model of actin alone. Amino acid residues that appear likely to contact tropomyosin are shown enclosed in white rectangles on the middle actin subunit; the bottom rectangle includes basic residues Lys-326, Lys-328, Arg-147, and the two others include Pro-333, Asp-25, and Arg-28. Basic amino acids in the Oda model are painted blue, acidic amino acids are red, and proline is pink. The pointed end of actin is facing up in each panel. (j) Enlargement of the actin subunit containing the white rectangles shown in f; residues noted above are labeled. (g–i) Three transverse sections of the reconstruction in panel a (indicated by triangles in a). The sections are viewed from the barbed end upward toward the pointed end of the filament with each section separated from the previous one by 27.5 Å (i.e., traversing 55 Å and equaling the length of actin monomers on either side of the filament); actin subdomains are numbered in each section. Tropomyosin on each side of these filament sections is fitted with a coiled-coil tropomyosin model (18), thus filling the elliptically shaped densities. Sectioned tropomyosin difference densities are superposed.

Figure 3

Evaluating electrostatic interactions between tropomyosin and F-actin. (a) Change in the electrostatic interaction energy values for tropomyosin repositioned on F-actin in the z-direction (plot marked by x) starting from the initial reference structure (0 point on the x axis of the plot), in this case holding the rotation and azimuth of tropomyosin fixed, and for tropomyosin rotated on F-actin (plot marked by o), in this case holding the z-position and azimuth constant. Positive values for the z-position change indicate longitudinal shifting of tropomyosin toward the pointed end of F-actin, and positive values for rotation indicate a turning of tropomyosin toward the open position on F-actin. Note the strong minima in each of the plots. The z-position and rotational shifts plotted in panel a represent 2D sections through the origin in the 3D plot in panel b. (b) A plot of the electrostatic energy landscape reflecting tropomyosin movement on F-actin as a function of both z-position and pseudo-rotation (0,0 reflects the z-position and rotation of tropomyosin on F-actin in the initial reference structure). Again note the energy minima.

Figure 4

Optimal electrostatic interactions between F-actin and tropomyosin. (a) Surface view showing the location of tropomyosin on F-actin that optimizes electrostatic interactions between the two structures. The rotation, azimuthal, and z directions discussed in the text are indicated by arrows. The pointed end of F-actin is facing up (as the arrow for z indicates) and actin subunits are numbered as in Table 1, tropomyosin quasi-repeat 4 is marked with a single asterisk and quasi-repeat 5 with a double asterisk (note that, by convention, the numbering of actin subunits and tropomyosin repeats do not match). (b and e) Segments of the F-actin-tropomyosin model in panel a are enlarged and rotated to highlight potential interactions of tropomyosin quasi-repeats 4 (b) and quasi-repeat 5 (e) with actin subunits. (c and f) The same as b and e but in wire/ribbon format. (d and g) The corresponding region of F-actin as in c and f, but here displaying the alignment of F-actin and tropomyosin models that were fitted into the F-actin-tropomyosin reconstruction (note that the relative positions of interacting amino acids correspond closely to those in c and f). The single arrow in g points to Lys-326, Arg-147, and Lys-328 residues of actin subunit 3 approaching Glu-181 on tropomyosin quasi-repeat 5, and the single arrow in d points to Arg-147 and Lys-328 of actin subunit 4 approaching residue Glu-139 on tropomyosin repeat 4. The double arrows point to actin Pro-333 on actin subunits 3 and 4 approaching Val-170 and Ser-132 on tropomyosin repeats 5 and 4, as well as to actin Asp-25 and Arg-28 close to Arg-167 and Glu-163 on tropomyosin repeat 5, and Lys-128, Arg-125, and Asp-121 on repeat 4. Each of the other tropomyosin quasi-repeats shows a similar but not identical pattern of interactions with respective actin subunits (not displayed; see Table 1). Basic amino acid residues in actin and tropomyosin are painted blue, acidic amino acids are red, and proline is coral. An identical grid search was performed to determine the optimal electrostatic interaction for tropomyosin with the recently published F-actin structure proposed by the Namba Laboratory (41). The z-position determined for tropomyosin on this model was the same as that found for the Oda model (22).

Figure 5

Relative location of tropomyosin and actin in the EM reconstruction of F-actin-tropomyosin. (a) Surface view of the reconstruction shown in yellow wire mesh to facilitate inspection of docked proteins. Here, both tropomyosin and F-actin models were fitted separately into the reconstruction using Chimera fitting tools (the atomic structure of just one of the docked actin subunits is displayed). (b–g) Cross-sections of actin viewed from the barbed end of F-actin (here, a was rotated by ∼180° before sectioning). Two cross-sections through regions of actin subunit 3 and tropomyosin repeat 5 are indicated by double-ended arrows in panel a and shown in b, c, e, and f. The outer boundaries of the docked actin and tropomyosin are shown as isosurfaces in b and e, and in more detail in wire/ribbon format in c and f. (b and c) Val-170, Arg-167, and Glu-163 on tropomyosin are seen oriented toward Pro-333, Asp-25, and Arg-28 on actin. (e and f) Glu-181 on tropomyosin is oriented toward Lys-326, Lys-328, and Arg-147 on actin. Note the perpendicular orientation of the tropomyosin elliptical axis with respect to the charged surface of actin. (This same arrangement is also seen over corresponding sections of the other tropomyosin quasi-repeats.) (d and g) The identical region of maps as in c and f, but here displaying the optimal alignment of F-actin and tropomyosin based on electrostatics, as in Fig. 4. Color-coding of amino acids is the same as in Fig. 4.