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. 2017 Jan 20;429(2):237-248.
doi: 10.1016/j.jmb.2016.11.030. Epub 2016 Dec 6.

Structural Basis of Arp2/3 Complex Inhibition by GMF, Coronin, and Arpin

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Structural Basis of Arp2/3 Complex Inhibition by GMF, Coronin, and Arpin

Olga S Sokolova et al. J Mol Biol. .
Free PMC article

Abstract

The evolutionarily conserved Arp2/3 complex plays a central role in nucleating the branched actin filament arrays that drive cell migration, endocytosis, and other processes. To better understand Arp2/3 complex regulation, we used single-particle electron microscopy to compare the structures of Arp2/3 complex bound to three different inhibitory ligands: glia maturation factor (GMF), Coronin, and Arpin. Although the three inhibitors have distinct binding sites on Arp2/3 complex, they each induced an "open" nucleation-inactive conformation. Coronin promoted a standard (previously described) open conformation of Arp2/3 complex, with the N-terminal β-propeller domain of Coronin positioned near the p35/ARPC2 subunit of Arp2/3 complex. GMF induced two distinct open conformations of Arp2/3 complex, which correlated with the two suggested binding sites for GMF. Furthermore, GMF synergized with Coronin in inhibiting actin nucleation by Arp2/3 complex. Arpin, which uses VCA-related acidic (A) motifs to interact with the Arp2/3 complex, induced the standard open conformation, and two new masses appeared at positions near Arp2 and Arp3. Furthermore, Arpin showed additive inhibitory effects on Arp2/3 complex with Coronin and GMF. Together, these data suggest that Arp2/3 complex conformation is highly polymorphic and that its activities can be controlled combinatorially by different inhibitory ligands.

Keywords: actin nucleation; conformation; single-particle EM; yeast.

Figures

Figure 1
Figure 1. Distribution of Arp2/3 complex conformations in ligand-bound and unbound states
(A) Two-dimensional projections of ‘standard’ open and closed conformations of ligand-free Arp2/3 complex. Upper row, representative class averages of open conformation. Bottom row, representative class averages of closed conformation. Scale bar, 10 nm. Arrowheads point to the cleft between Arp2 and Arp3 in the open conformation. Cartoon panels in each row show the 7 subunits of Arp2/3 complex with color-coding as in all other figures: Arp2, red; Arp3, orange, ARPC1/p40, green, ARPC2/p35, blue; ARPC3/p21, light purple; ARPC4/p19, dark purple; ARPC5/p15, yellow. (B) Percentage of Arp2/3 complex particles in the open (inactive) versus closed (primed for nucleation) conformations (Arp2/3, n = 262; Arp2/3+Gmf1, n = 731; Arp2/3+Arpin, n = 1130; Arp2/3+Crn1, n = 362). (C) Two-dimensional projections of GST-Crn1 bound to Arp2/3 complex (left). Arrows highlight the visible new masses, which likely represent the globular β-propeller domains of a Crn1 dimer. Scale bar, 10 nm. Cartoon (right) depicts proposed arrangement of two Crn1 molecules (dark blue) dimerized by GST (green dots), with one Crn1 molecule in the dimer interacting with the p35/ARPC2 subunit of Arp2/3 complex.
Figure 2
Figure 2. Three-dimensional structures of Gmf1-bound Arp2/3 complexes
(A) Two-dimensional projections of Gmf1-bound Arp2/3 complex. Upper row, from left to the right: re-projection of the crystal structure of Arp2/3-GMF complex [14], free Arp2/3 2D projection in the same orientation, two representative 2D projections of Arp2/3-Gmf1 in standard open conformation, and two 2D projections of Arp2/3-Gmf1 in new open conformation. Numbers on the right reflect % of the particles in each class. Bottom row: difference maps, generated by subtracting the 2D projection of free Arp2/3 from Arp2/3-Gmf1 projections, respectively. White arrowheads highlight adding of the new mass, orange arrowheads highlight loss of the mass. (B) Crystal structure of GMF-bound Arp2/3 complex [14] filtered to 25 Å resolution using UCSF Chimera [51]. Arp2/3 complex subunits, color-coded and labeled as in Figure 1A. Arrows highlight the position of GMF (grey) bound to Arp2 and p40/ARPC1 subunits. (C,D) Three-dimensional reconstructions of Gmf1-bound Arp2/3 complex in the standard open conformation (C) and the new open conformation (D). Additional densities, approximately the size of Gmf1, are shaded in pink and labeled with arrows. Asterisk in (C) marks a new mass at the proposed second binding site for GMF on Arp3. Three separate views of Arp2/3 complex shown in B-D are aligned to each other for comparison. Scale bar, 10 nm.
Figure 3
Figure 3. Gmf1 and Crn1 synergize in inhibiting Arp2/3-mediated actin assembly in vitro
(A, B) Bulk actin polymerization assays containing G-actin (2 µM, 5% pyrene-labeled) and the indicated concentrations of Arp2/3 complex, VCA, Gmf1, and Crn1. (C) Bulk actin polymerization assays, performed as in A and B, except using full-length Las17 (yeast WASP) instead of VCA. (D) Percentage of Arp2/3 complex particles with Crn1 bound in the presence and absence of Gmf1 (- Gmf1, n = 366; + Gmf1, n = 398).
Figure 4
Figure 4. Three-dimensional structure of Arpin-bound Arp2/3 complex
(A) Two-dimensional projections of Arpin-bound Arp2/3 complex. Arrows highlight new masses, which likely represent the globular core domains of Arpin [40]. Yellow arrows indicate the presence of a single new mass, while white arrows indicate two new masses. Scale bar, 10 nm. Lower panel includes corresponding cartoons of each class average. The larger cartoon on the right shows Arp2/3 complex with superimposed positions of Arpin (filled circles). Dashed circles have a 5 nm radius, corresponding to the estimated length connecting the center of Arpin globular domain and its C-terminal acidic (A) motif. The circles were used to map potential Arpin-binding sites on Arp2/3 complex (open circles). (B) Three-dimensional reconstruction of Arpin-bound Arp2/3 complex viewed from different angles. Additional density, attributed to Arpin, is shaded pink. (C) Crystal structure of Arp2/3 complex [14] filtered to 25 Å resolution using UCSF Chimera [51], with subunits color-coded and labeled. Views are aligned with those in B for comparison. Scale bar, 10 nm. (D) Bulk actin polymerization assays containing G-actin (2 µM, 5% pyrene-labeled) and the indicated concentrations of Arp2/3 complex, VCA, Arpin, Gmf1, and Crn1.

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