Structures of the free and capped ends of the actin filament

Abstract

The barbed and pointed ends of the actin filament (F-actin) are the sites of growth and shrinkage and the targets of capping proteins that block subunit exchange, including CapZ at the barbed end and tropomodulin at the pointed end. We describe cryo-electron microscopy structures of the free and capped ends of F-actin. Terminal subunits at the free barbed end adopt a "flat" F-actin conformation. CapZ binds with minor changes to the barbed end but with major changes to itself. By contrast, subunits at the free pointed end adopt a "twisted" monomeric actin (G-actin) conformation. Tropomodulin binding forces the second subunit into an F-actin conformation. The structures reveal how the ends differ from the middle in F-actin and how these differences control subunit addition, dissociation, capping, and interactions with end-binding proteins.

Fig. 1:. Free barbed end.

(A) Schematic representation of the G- to F-actin transition. A scissor-like ~20° rotation of the outer domain (subdomains 1 and 2) relative to the inner domain (subdomains 3 and 4) produces a flatter conformation of subunits in F-actin. (B) Cryo-EM map of the free barbed end at 3.30-Å resolution. The terminal and penultimate subunits are shown in two different shades of blue. (C) The terminal and penultimate subunits (blue) adopt the classical F-actin conformation of subunits in the middle of F-actin (grey). Subunits were superimposed based on the inner domain (surface representation) to highlight differences in orientation of the outer domain (ribbon representation). (D) Fit to the cryo-EM map of the W-loop of the terminal subunit (blue) vs. a subunit from the middle of the filament (grey). (E) In the middle of F-actin, the W-loop and the C-terminus of actin form a pincer-like structure that grasps the D-loop of the subunit below (grey and cyan, respectively). This interaction is absent for subunits at the barbed end (blue), resulting in the W-loop adopting a G-actin conformation and the C-terminus moving by 2.0 Å (red arrows).

Fig. 2:. CapZ-capped barbed end.

(A) Domain diagram of CapZ subunits α and β. (B) Mushroom-like structure of CapZ and transition between filament-bound and unbound states. In filament-bound CapZ, the mushroom head flattens and the tentacles engage the hydrophobic clefts of barbed end subunits. (C) Cryo-EM map of the CapZ-capped barbed end at 2.79-Å resolution, showing the terminal and penultimate actin subunits in two shades of blue and CapZ α and β in pink and magenta, respectively. (D) Splaying of the short-pitch pair formed by the terminal and penultimate subunits (blue) as compared to a short-pitch pair from the middle of F-actin (grey). The short-pitch pairs were superimposed based on the penultimate subunit to highlight splaying of the terminal subunit, showing a maximum displacement of 1.5 Å resulting from a rotation of ~2° (red arrow). Splaying is likely favored by missing contacts of the inter-strand plug at the barbed end compared to the middle of F-actin (dashed red curves). (E) Comparison of the filament-bound (pink, magenta) and unbound (22) (grey, PDB code: 3AA7) structures of CapZ. The superimposition, based on CapZα, highlights a ~15° rotation of CapZβ (red arrow) that results in flattening of the mushroom head. (F) The actin-binding surface of CapZ consists mostly of two antiparallel helices and the α and β tentacles. Comparison of the filament-bound (pink, magenta) and unbound (grey) structures shows that the helices contain π-bulges that change conformation between these two states (red arrows). The tentacles, which are disordered in the unbound structure, project out in the filament-bound structure to engage the two barbed end subunits. (G) Close-up view CapZ’s interaction with the barbed end, with the binding interface colored pink and magenta. CapZ residues participating in the interaction are shown and labeled in Fig. S9C.

Fig. 3:. Free pointed end.

(A) Cryo-EM map of the free pointed end at 2.84-Å resolution, showing the first and second subunits in two different shades of green. The D-loop of subunits at the pointed end is disordered. (B) The first (left) and second (right) subunits at the pointed end adopt a G-actin conformation, where the outer domain is rotated ~20° (red arrow) compared to subunits in the middle of the filament (grey). Subunits were superimposed based on their inner domains (surface representation) to highlight differences in orientation of their outer domains (ribbon representation).

Fig. 4:. Tmod-capped pointed end.

(A) Domain diagram of Tmod, comprising alternating tropomyosin (TMBS1, TMBS2, disordered in the structure) and actin (ABS1, ABS2) binding sites. (B) Cryo-EM map of the Tmod-capped pointed end at 3.26-Å resolution, showing the first and second subunits in two shades of green and Tmod in orange. The view on the right is approximately down the longitudinal axis of F-actin and shows a ribbon representation of Tmod. ABS1 caps the first actin subunit whereas ABS2 wedges into a cleft formed by the first three actin subunits. Most of the interactions with actin are mediated by the b-strand to a-helix loops of the leucine-rich repeat (LRR) domain of ABS2. Tmod residues involved in the interaction are shown and labeled in Fig. S9D. (C) The first actin subunit (left) adopts a G-actin conformation with the outer domain rotated by ~20° (red arrow) compared to subunits in the middle of F-actin (grey). The second actin subunit (right) adopts an F-actin conformation. Subunits were superimposed based on their inner domains (surface representation) to highlight differences in orientation of their outer domains (ribbon representation).

Fig. 5:. Model of subunit association/dissociation at the free and capped ends of F-actin.

Structures described here show that subunits in F-actin have different conformations depending on whether they are in middle or at the ends of F-actin, and different from G-actin. Importantly, these are not nucleotide-dependent conformational differences, which are relatively minor in both G- and F-actin (see text). The conformational differences at the ends of F-actin correlate with the association/dissociation constants of subunits at the ends of F-actin. Only the main pathway at equilibrium is depicted, with ATP-actin preferwentially adding to the barbed end and ADP-actin dissociating from the pointed end (see Ref. for other possible reactions). Structural differences explain the asymmetric association of ATP-actin monomers to the barbed and pointed ends of F-actin, with ATP-bound monomers being more likely to undergo the G- to F-actin transition required for preferential binding to the barbed end than ADP-bound monomers. CapZ and Tmod inhibit subunit exchange at the barbed and pointed ends, respectively, by structrual mechanisms revealed in this study.