Mechanism of synergistic actin filament pointed end depolymerization by cyclase-associated protein and cofilin

Figures

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  Fig. 1. The crystal structure of the HFD domain of CAP in complex with ADP-actin and ADF-H domain. a The HFD domain of CAP (magenta) binds to the pointed end of an ADP-actin monomer (green) between subdomains 2 and 4, whereas twinfilin’s ADF-H domain (cyan) binds to the barbed end of the monomer between subdomains 1 and 3. b Highlighted in yellow are actin’s D-loop and the adjacent α-helix (residues 55–61), which are spatially coordinated by Tyr162 and Phe163 of the HFD domain. Lys50 and Glu57 of actin form salt bridges with Glu119 and Arg118 of the HFD domain, respectively. Met47 of actin fits into a hydrophobic cavity formed by a kink in the contacting α-helix of the HFD domain. Selected residues are presented in their corresponding 2F0 – FC (σ = 1.0) electron density map. c The HFD domain covers an extensive binding surface of 1208 Å2 (yellow) predominantly on actin subdomain 2. d Different conformational states of actin monomers analyzed by comparing the twist between actin structures superimposed on their inner domains (subdomains 3 and 4) and measuring the angle of the outer domain (subdomain 1) relative to the ADP-state of F-actin (PDB = 6djo). The HFD domain-bound actin monomer…

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  Fig. 2. CAP catalyzes pointed end depolymerization of cofilin-decorated actin filaments. a The actin monomers in a cofilin-decorated filament (PDB = 5yu8) display a similar conformation to the one in our crystal structure (Fig. 1d), allowing docking of two HFD domains of CAP to the two pointed end actin subunits without steric clashes. Rotated view shows that the N-termini of the two HFD domains (Ala39) are within 5 nm, suggesting the filament-bound CAP can still oligomerize. b A schematic of the actin filament pointed end depolymerization assay. c Representative kymographs showing the pointed end depolymerization of cofilin-decorated filaments in the absence (top) and presence (bottom) of 3 μM N-CAP. d The pointed end depolymerization activity of N-CAP fitted by first-order saturation suggests a Kd of 0.978 + 0.148 μM for N-CAP binding to the pointed end of cofilin-decorated actin filament. Error bars, S.D., n ≥ 5 for each point. e Examples of actin filament reannealing experiments, where actin filaments labeled with different colors were mixed and imaged. f Quantification of the reannealing experiments performed in the absence of N-CAP (control) and presence of wild-type N-CAP or a…

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  Fig. 3. Conformation of the bare filament pointed end is not favorable for binding of CAP. a Superimposition of ADP-actin from the HFD domain/ADF-H domain/actin structure to the pointed end of a bare ADP-actin filament (PDB = 6djo) by aligning subdomains 3 and 4 of actins causes clashes between the D-loop of actin and the HFD domain. As a result of nonoptimal docking, the HFD domain bound to the penultimate actin monomer collides with the ultimate actin monomer in the filament. Steric clashes are indicated with red spheres. b Superimposition of ADP-actin from the HFD domain/ADF-H domain/actin structure to the pointed end of a bare actin filament by aligning subdomain 1 of actins does not cause clashes between actin and the HFD domain. However, the conformation of bare filamentous actin (green) is not optimal for HFD domain binding when compared to the crystallized actin conformation (orange). This is due to the difference in the twist of the actin (see Fig. 1d) that decreases contacts between the HFD domain and actin subdomain 4. c Examples of bare actin filament pointed end depolymerization assays (by single-filament microfluidics approach) performed in the absence and presence of 30 μM…

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  Fig. 4. Molecular mechanism of actin filament pointed end depolymerization by CAP. a Energy-minimized model of two HFD domains bound to a cofilin-decorated actin filament pointed end. HFD domains bind actin through two interfaces: The one determined in our crystal structure (interface 1, yellow), and interface 2 (orange), which forms between the ultimate actin (actin A) and the HFD domain bound to the penultimate actin (actin B). Residues shown in sticks were mutated to validate the binding interfaces. b Cofilin-decorated actin pointed end depolymerization rates facilitated by N-CAP mutants as determined by single filament microfluidics. n ≥ 5 for each concentration. c Different CAP constructs used in this study. The dimeric HFD domain lacks the oligomerization domain (OD), but was fused to glutathione-S-transferase (GST) from its C-terminus to form dimers (see Supplementary Fig. 4). PP=polyproline region, DM=dimerization motif. d Pointed end depolymerization rates of cofilin-decorated actin filaments facilitated by different CAP constructs. The isolated HFD domain is inefficient in depolymerizing cofilin-decorated actin filaments at low concentrations, whereas dimerization of this…

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  Fig. 5. CAP destabilizes the interface between the terminal actins at the filament pointed end. a All-atom molecular dynamics simulations of cofilin-decorated actin filaments (consisting of four actins and three cofilins; see Supplementary Fig. 5a) in the absence of CAP, and in the presence of two HFD domains bound to filament pointed ends. MD simulations show that the interface between terminal actins of the filament opens in the presence of HFD domains, as indicated by the decrease in the number of contacts between these subunits. b The number of contacts between actins A and B at the pointed end from three simulations (started from three independently generated configurations; see Methods) shown as a function of time. Left: Cofilin-decorated ADP-actin pointed end. Right: With two HFD domains bound to the cofilin-decorated ADP-actin pointed end. The number of contacts was defined as the number of residues from both chains within 3 Å of each other. c The contact frequencies of individual residues between actin A and actin B are shown. Left: The cofilin-decorated ADP-actin filament pointed end. Right: With two HFD domains bound to cofilin-decorated ADP-actin filament pointed end. The…

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  Fig. 6. Mechanism of actin monomer recycling by CAP. a The C-terminal half of CAP is composed of CARP and WH2 domains and catalyzes ADP-to-ATP nucleotide exchange on actin monomers. The HFD domain (magenta) binds to an actin monomer (blue) using a different interface compared to the dimeric CARP domain of CAP (orange/yellow), which binds to the backside of actin monomers. A small steric clash between the CARP domain and ADF-H domain (cyan) is indicated with an arrow. b Based on prior structural data on WH2 domains44, mutagenesis34 and atomistic molecular dynamics simulations34, the WH2 domain of CAP binds to actin using an interface that overlaps with the ones of the HFD and ADF-H domains (light gray). c Gel filtration assay showing the formation of a tripartite complex between HFD domain, ADF-H domain, and ADP-G-actin (upper panel). Line profiles present the abundance of the proteins in each gel filtration fraction as analyzed by SDS-PAGE, and the gray area corresponds to the elution profile. Upon addition of the C-terminal half of CAP (C-CAP), the HFD and ADF-H domains are released from actin, and a complex between C-CAP and the actin monomer forms (lower panel). n = 3; error bars…

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  Fig. 7. A working model for actin filament depolymerization and monomer recycling by CAP. The different states of the model are based on prior structural data34,43,44, as well as crystal structure and atomistic molecular dynamics models from this study. The CARP domain dimer34 (yellow/orange) is connected by Rosetta-modeled linkers to the HFD domains (magenta). Please note that for simplicity CAP is presented as a dimer in the model. (1) ADP-actin filaments (blue/green) are severed by cofilin (cyan). (2) CAP binds with high affinity to the cofilin-decorated filament pointed end through its two HFD domains, and destabilizes the interface between the two terminal subunits of the actin filament. (3) The ultimate ADP-actin monomer dissociates from the filament pointed end, due to spatial restrains and lost contacts to the penultimate monomer. The HFD domain and cofilin stay associated with the actin monomer. (4) The freshly depolymerized actin monomer is handed over from the HFD domain to the CARP domain of CAP, while the second HFD domain remains bound to the actin filament pointed end. (5) The WH2 domain of CAP wraps around the actin monomer, and together with the adjacent CARP domain…