Molecular mechanism of Arp2/3 complex inhibition by Arpin

Figures

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  Fig. 1. Arpin binds to a single site on subunit Arp3 of Arp2/3 complex. a Comparison of the domains and sequences of N-WASP (NPF) and Arpin (inhibitor), both of which contain C-terminal CA regions (UniProt accession codes: human N-WASP, O00401 and human Arpin, Q7Z6K5). b–d Comparison of cryo-EM maps (and ribbon diagrams) low-pass filtered at the 5-Å resolution of Arp2/3 complex alone15 (b) and with bound Arpin (c) and N-WASP CA7 (d). Arp2/3 complex subunits are labeled in part b, and colored as follows: Arp2, cyan; Arp3, green; ArpC1, yellow; ArpC2, gray; ArpC3, blue-white; ArpC4, blue-white; ArpC5, gray. Arpin and N-WASP are colored marine blue and magenta, respectively. Note that the Arpin_CA and N-WASP CA densities on Arp3 are very similar. Panels b–d were generated with the program Chimera41, and the maps were contoured at 4σ. e–h ITC titrations of full-length Arpin or Arpin_CA (in the syringe) into Arp2/3 complex (in the cell) in the ATP or ADP-bound states (as indicated). For each titration, the experimental conditions, including temperature (T) and protein concentrations, and the fitting parameters, including stoichiometry (N), dissociation constant (Kd), and goodness of the fit…

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  Fig. 2. Cryo-EM structure of Arpin_CA bound to Arp2/3 complex. a Cryo-EM map of Arpin_CA bound to Arp2/3 complex colored by resolution (as indicated by the side bar). Labels indicate the approximate location of Arp2/3 complex subunits. b Representative region of the 3.24-Å resolution cryo-EM map, showing the definition of side chains for the α-helix formed by residues Y253-A268 of ArpC2. c Cryo-EM map of Arpin_CA (marine blue) bound to Arp2/3 complex. Arp2/3 complex subunits are labeled and colored as follows: Arp2, cyan; Arp3, green; ArpC1, yellow; ArpC2, gray; ArpC3, blue-white; ArpC4, blue-white; ArpC5, gray. The Arpin_CA density appears as two separate patches shown in insets and corresponding to the C-helix and A domain.

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  Fig. 3. Interactions of Arpin and N-WASP with Arp2/3 complex. a, b Superimposition of Arpin_CA-Arp3 (marine blue and green) onto N-WASP-Arp2 (a) and N-WASP-Arp3 (b) (magenta and gray). c The contact surface (orange) of Arpin on Arp3 involves Arpin’s C-helix and the A domain, whereas the negatively charged linker between these two regions is flexible and does not participate in specific contacts. d–g Comparison of the specific interactions of the A domain (d and f) and C-helix (e and g) of Arpin and N-WASP with Arp3 (green with orange side chains). Insets show details of the interactions of the C-helices of Arpin (e) and N-WASP (g) with the C-terminal tail of Arp3. The inset in part e shows a comparison of the C-terminal tail of Arp3 in both structures (green-orange, Arpin structure; gray, N-WASP structure.

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  Fig. 4. Replacing the C-helix of Arpin by that of N-WASP turns Arpin into an NPF. a Domain diagrams of human Arpin and N-WASP (top). A dashed box highlights the C-terminal region included in the sequence alignment shown at the bottom. The alignment includes Arpin sequences from several species (upper portion) and several human NPFs (lower portion). UniProt accession codes are included with the name of each sequence. The middle portion of the alignment shows the human Arpin constructs and hybrid constructs of N-WASP and Arpin used in this study. Amino acids added or mutated relative to wild-type Arpin are boxed. b Left, time-course of actin polymerization by Arp2/3 complex activated by N-WASP WCA (protein concentrations are indicated), with and without Arpin constructs (10 µM). Data are shown as the average curve from three independent experiments with s.d. error bars in lighter color. Insets show a zoom of experimental curves that overlap. Right, maximum polymerization rates calculated from experiments analogous to those shown on the left (n = 3) and performed at three different concentrations of the Arpin constructs (as indicated, see also Supplementary Fig. 4a). c Left, time-course of…

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  Fig. 5. Arpin C-helix mutants impair Arp2/3 complex binding and cell migration. a Immunoprecipitation of Arp2/3 complex subunits ArpC2 and ArpC3 from HEK-293T cells expressing Flag-tagged Arpin WT and mutants W195D and I199D/M200D. Right panel shows the loading controls of cell extracts and reveals both endogenous and exogenously expressed Arpin. b Western blot detection of Flag-tagged Arpin WT and mutants W195D and I199D/M200D expressed in ARPIN −/− stable MCF10A cell lines (loading control, α-tubulin). Arpin is not detected in control ARPIN −/− cells extracts. Uncropped and unprocessed blots are provided as a Source Data file. c Migration persistence of control parental and ARPIN −/− MCF10A cells compared to those of ARPIN −/− cells expressing constructs Arpin WT and mutants W195D and I199D/M200D (raw migration data in Supplementary Fig. 5c). The statistical significance of the measurements was calculated using a one-way ANOVA test (exact P-values are listed in the figure). Source Data are provided as a Source Data file. N = 74 cells for MCF10A parental, ARPIN −/− and WT cl.1, N = 72 cells for WT cl.2, N = 75 cells for W195D cl.1, N = 69 cells for W195D cl.2, N = 76 cells for…

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  Fig. 6. Model of Arp2/3 complex inhibition by Arpin versus activation by actin—NPF. Arp2/3 complex is activated by actin—NPF binding to two sites, a high-affinity site-1 on Arp2-ArpC1 and a lower affinity site-2 on Arp37 (Activation, top). Actin-NPF binding to site-1 shifts the equilibrium towards the short-pitch conformation, which also facilitates actin—NPF binding to Arp3. Arpin inhibits Arp2/3 complex activation through a combination of two effects (Inhibition, bottom). By locking the inhibitory tail of Arp320, Arpin inhibits the short-pitch transition that actin—NPF binding to site-1 promotes. By binding to Arp3, Arpin directly competes with actin—NPF binding to site-2.