Mechanism of actin filament nucleation by the bacterial effector VopL

Abstract

Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected into eukaryotic host cells. VopL contains three N-terminal Wiskott-Aldrich homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own and requires the VCD for activity. The VCD forms a U-shaped dimer in the crystal, stabilized by a terminal coiled coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data lead to a model in which VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.

Figure 1

Both the WH2 motifs and VCD contribute to actin nucleation by VopL. (a) Domain structures of VopL constructs used in this work. All constructs lack the N-terminal secretion sequence needed for VopL translocation into host cells, whose deletion does not affect activity (not shown). P: Proline-rich motif. WH2: WASP Homology 2 domain. VCD: VopL C-terminal domain. GGS: (Gly-Gly-Ser) linker. The half-time (t_1/2) of actin assembly for each VopL protein is given by the average and standard deviation measured in three experiments. Assays contained 50 nM or 5 nM VopL proteins, as indicated. (b) Actin assembly assays contained the indicated concentrations of VopL constructs W₃-C, W₃ or W₄. (c) Actin assembly assays contained 50 nM W₃-C, W₂-C, W₁-C or VCD. Figures in this manuscript were all generated using Adobe Illustrator CS2.

Figure 2

Dimerization is necessary for actin nucleation by the VopL WH2 motifs. (a) Size exclusion chromatography—multi-angle laser light scattering (MALLS) analysis of VopL constructs W₃-C (red) and VCD (blue). Normalized scattered light in the chromatographic elution (right y-axis) is superimposed on the molecular weight distribution (left y-axis). (b) MALLS analysis of VCDΔh. Data plotted as in (a). (c) Actin polymerization assays performed with 4 μM actin and 50 nM W₃-C, W₂-C, W₁-C, VCD, W₃-GST, W₂-GST or W₁-GST.

Figure 3

Structure of the VCD dimer. (a) Ribbon diagram of the structure; monomers are colored blue and green. The N- and C-termini of each monomer are indicated. Red dashed lines indicate regions not observed in the electron density map for the green monomer (analogous regions not shown in the blue monomer). Boxed regions are enlarged in (b) and (c), which show detailed views of dimer interface. Black dashed lines indicate hydrogen bonds. In all panels sidechains of residues discussed in the text are shown as sticks.

Figure 4

The VCD contributes to actin assembly activity by dimerization and contacts to actin. Assays were performed with 4 μM actin and 50 nM (a, b) or 5 nM (c) of the indicated VopL proteins. (a) Comparison of W₃-C, W₃-CΔh and W₃. (b) Comparison of W₃-C with the VCD arm mutants, W₃-C_KRR and W₃-C_DVP. (c) Comparison of W₃-C with the VCD base mutants, W₃-C_KYR, W₃-C_YR and W₃-C_EDE.

Figure 5

Increasing the linker between WH2c and the VCD increases actin assembly activity. Assays contained of each 50 nM VopL protein, W₃-C, W₂-C, W₁-C, W₂-L-C or W₁-L-C.

Figure 6

Model for a minimal actin nucleus assembled by VopL. VCD is indicated by blue blocks with ribbons representing the coiled coil. Initial actin monomers are indicated by yellow boxes. Actin-bound WH2c motifs are shown as red cylinders. Red lines indicate linkers between WH2c and VCD. Additional actin monomers assembled by WH2a and WH2b would bind to the upper surfaces of the WH2c-bound actins, making additional short- and long-pitch contacts, further stabilizing the assembly. Actins are implicitly shown with barbed ends directed away from the VCD, but our current data do not speak to the orientation of actin monomers in the VopL nucleus.