A. Sequence alignment of WASP and N-WASP VCA C regions and the fifth repeat element of EspFU. Helix residues in the autoinhibited GBD-C structure and GBD-R33 complex are blue. Residues in the extended EspFU arm are red. Aligned hydrophobic residues are boxed in yellow. EspFU residues that contact the GBD are indicated by •. Sites of EspFU mutations used in panels B and D are indicated by *. Proline-rich motif is boxed in gray. The C-termini of EspFU single repeat constructs used throughout this work are indicated below the sequence. B. Pyrene-actin fluorescence measured during assembly of 4 µM actin (5% pyrene-labeled) plus 10 nM Arp2/3 complex (black) plus 25 nM N-WASPC (blue) plus 500 nM of: R47 (green), R33 (red), R18 (orange), R14 (cyan), R33**L (pink ) or R33*** (V4A/L8A/L12A, grey). C. Concentration of filament barbed ends produced during assembly of 4 µM actin by 10 nM Arp2/3 complex, 25 nM N-WASPC and increasing concentrations of R47 (black squares; red curve shows fit to single site binding isotherm) or 500 nM Cdc42-GMPPNP (green triangle). Blue circle shows barbed ends produced by actin plus Arp2/3 plus 25 nM N-WASP VCA. D. For EspFU proteins listed, table shows free energy of unfolding of GBD-EspFU fusion, dissociation constant for binding to N-WASPC, and actin filament barbed ends produced by assays in panel B. In “none” row, ΔGu represents melting of the isolated GBD, B.E. represents assays performed with only N-WASPC and Arp2/3 complex.