Mechanisms of disassembly at the actin filament pointed and barbed ends

Figures

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  Fig. 1.. Cofilactin structure. (A) Cryo-EM map of cofilactin at ~2.1 Å resolution (actin, gray; cofilin, yellow). (B) Inset showing Mg2+-ADP and water molecules in the nucleotide-binding cleft. A secondary inset highlights the Mg2+-binding site. (C) Inset showing interactions of cofilin (yellow) with two actin subunits along the long-pitch helix (gray transparent surface with Cα traces in gray and blue). A secondary inset highlights cofilin’s helix L111-K127 and its N terminus bound in the H-cleft of the actin subunit above (toward the pointed end). Residue S3, whose phosphorylation inhibits cofilin binding and severing, is shown in red. (D) Three perpendicular views of superimposed Cα traces illustrating how actin’s conformation in the cofilactin structure (blue, this work) resembles that of other G-actin structures (gray; PDB: 1J6Z, 2PBD, 3A3Z, 1KXP, 4PKG, 2A42, 1IJJ, 1Y64, and 4M63) and differs from F-actin (red; PDB: 8F8P). Structures were superimposed on the inner domain (residues 145 to 336) to highlight outer-domain rotation. Cofilactin, however, differs from both G- and F-actin in having a more open H-cleft, as the binding of cofilin’s helix and N terminus separates subdomain-1 of…

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  Fig. 2.. Pointed-end structures with cofilin, CAP, or both. (A) Domain diagram of CAP (OD, oligomerization domain; PR, proline-rich; WH2, WASP-homology-2). (B to F) Cryo-EM maps of the pointed end with bound cofilin (yellow), one HFD (orange), two HFDs, and cofilin with one or two HFDs, respectively (resolutions indicated). The first and second actin subunits are dark and light green, respectively. (G and H) The two pointed-end subunits with bound cofilin or HFD adopt a twisted G-actin–like conformation, similar to the free pointed end and distinct from the flat mid-filament F-actin conformation (gray) (7). The F- to G-actin transition involves a ~20° rotation of the outer domain relative to the inner domain (3). (I) Binding of the second HFD generates a steric clash with the first actin subunit, resolved by a ~4.0 Å lateral displacement (red arrow). The displacement is illustrated by comparison of the first subunit in the one-HFD (gray) structure. (J and K) Top and side views show that the cofilin-induced undertwist of the short-pitch helix amplifies this clash, increasing the lateral displacement of the first subunit to ~7.6 Å. The first subunit moves with its bound cofilin and HFD and…

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  Fig. 3.. Barbed-end structures with CP, cofilin, or both. (A to F) Cryo-EM maps of the barbed end with bound CP (CPα, magenta; CPβ, pink), cofilin (yellow), and structures of CP with cofilin or cofilin alone. (G) In all cofilin-containing structures, the two barbed-end subunits (dark and light blue) adopt a twisted G-actin–like conformation distinct from the flat conformation of mid-filament subunits, the free barbed end, or the CP-bound barbed end (gray) (7). (H) Bottom-view showing how the cofilin-induced undertwist of the short-pitch helix rotates CP, which weakens its interaction with the barbed end. (I) CP retains its barbed-end–bound conformation (7), independent of cofilin binding and of the F- to G-actin transition of barbed-end subunits, distinct from the more curved conformation of CP’s mushroom head in the unbound state (gray) (39). (J) CP β-tentacle (magenta in transparent cryo-EM map density) inserted into the H-cleft of the last subunit (dark blue map) from part A. (K) Left to right, position of the α-tentacle in the structures from parts (A) to (C), showing progressive displacement by cofilin, first due to the F- to G-actin transition and undertwist and then through direct…