The structure of the actin filament uncapping complex mediated by twinfilin

Figures

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  Fig. 1. The twinfilin/CP/actin complex. (A) Front view of the pentameric complex in cartoon representation. CP consists of two subunits, α-subunit (CPα) and β-subunit (CPβ). Twinfilin comprises two ADF-H domains (D1 and D2), a linker between D1 and D2 that includes a helix (residues 151 to 165), and a C-terminal tail (Tail; residues 316 to 342, the last eight amino acids are disordered). Actin subunit 1 is bound to twinfilin D1, and subunit 2 to twinfilin D2. The twinfilin secondary structure elements are colored differentially, helices in red, strands in cyan, and loops and extended regions in orange, with the Tail in lime green. (B) Front view [same as in (A)], in which the actin subunits and twinfilin are represented as surfaces. Twinfilin is shown entirely in orange. (C and D) Back and side views, respectively.

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  Fig. 2. Binary interactions in the twinfilin/CP/actin complex. (A) Front and back views of actin bound to twinfilin D1 or twinfilin D2. (B) Two views of the CP interaction with actin, in which actin 1 and actin 2 are shown in similar orientations. (C) Three orientations of the CP/twinfilin interaction. Examples of the electron density of key features are shown in fig. S5 (H to K).

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  Fig. 3. Conformations of CP. (A) Comparison of the twinfilin tail (yellow) binding site with CARMIL (cyan) on CP. Both CP-binding peptides run in the same direction, and N terminus of the twinfilin tail is labeled N. (B) The actin-binding site on the CP α-tentacle is distant from the twinfilin tail-binding site. (C and D) Structural superimposition of the β-subunits of CP reveals that the conformation of CP in the twinfilin/CP/actin complex is different to the CARMIL-bound and unbound conformations of CP. (E) Superimposition reveals that the conformation of CP in the twinfilin/CP/actin complex is similar to the V-1–bound conformation of CP. α- and β-subunits of CP are colored light green and dark green, respectively, for the CARMIL, unbound, and V-1 complexes. Black arrows indicate conformational changes in CP in adopting the twinfilin/CP/actin complex structure.

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  Fig. 4. Comparison of the CP-binding mode to Arp1 in the dynactin complex (Arp1/CP) with the binding mode to actin in actin/twinfilin/CP complex. (A and B) Focus on the β- and α-tentacles, respectively, which are highlighted by black circles. The β-tentacle is disordered and unbound in the actin/twinfilin/CP complex, and the α-tentacle is partially bound and ordered, relative to the dynactin complex. Enlargements of the tentacle regions show the superimpositions of the CP (α- and β-subunits colored red and blue, respectively) from the dynactin complex on to actin (green and teal) and twinfilin (yellow) from the actin/twinfilin/CP complex. (A) Enlargement, the twinfilin linker (yellow) binds to the β-tentacle–binding site on actin 2 (green). (B) Enlargement, the N terminus of twinfilin D1 (yellow) occupies half of the α-tentacle–binding site on actin 1 (teal).

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  Fig. 5. V-1 uncaps filament barbed ends in the presence of twinfilin. Pyrene-actin polymerization assay showing uncapping of F-actin seeds by V-1 in the presence of twinfilin. Unlabeled F-actin seeds (0.5 μM) were capped with 100 nM CP and subsequently polymerized in either 2 μM actin (10% pyrene)/2.8 μM profilin (red) or 2 μM actin (10% pyrene)/2.8 μM profilin supplemented with either 1 μM twinfilin (yellow), 5 μM V-1 (purple), or a mixture of 1 μM twinfilin and 5 μM V-1 (blue). As a control, F-actin seeds were polymerized in the absence of CP (black). As shown in the profile, the presence of both twinfilin and V-1 induces accelerated polymerization of precapped F-actin seeds (blue) to almost the same level as the positive control (black). By contrast, twinfilin alone (yellow) induces very low level of polymerization, similar to CP alone (red), while V-1 alone (purple) only induces minimal polymerization. AU, arbitrary units.

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  Fig. 6. Models of the filament uncapping. (A) Cartoon comparison of CP and CP/twinfilin association at the barbed end of a filament. (B) Twinfilin-aided uncapping is a result of twinfilin ADF-H domains inducing G-actin–like conformations in the terminal two actin protomers, weakening actin:actin interactions in the filament, leading to the dissociation of the complex. (C) Twinfilin/V-1–aided uncapping requires space for V-1 to reach its binding site on CP via a wobble state of the twinfilin-bound complex. Once V-1 is bound, CP is unable to reassociate with actin. In vitro effects of twinfilin alone on actin filaments and comparisons of uncapping models in the absence of twinfilin are shown in fig. S9.