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 actin polymerization by Arp2/3 complex activated by N-WASP WCA or hybrid N-WASP-Arpin constructs. The data are shown as described in part b, and the concentrations of the proteins are indicated. Right, maximum polymerization rates calculated from the experiments shown on the left (n = 3). For parts b and c, the statistical significance of the measurements was calculated using an unpaired, two-tailed t-test (P-values listed in the figure). The source data are provided as a Source Data file. d ITC titration of construct Hybrid_WH2_C-helix into Arp2/3 complex. The experimental conditions and fitting parameters are listed. Hybrid_WH2_C-helix is the only hybrid construct that activates Arp2/3 complex, and like N-WASP WCA binds to two sites on Arp2/3 complex.