Figure 1.
Auto-inhibited WASP is activated by binding Cdc42, and can then work with the Arp2/3 complex to nucleate new actin filaments (direction indicated by a dashed block arrow). By forming 1∶1 complexes with G-actin, ABPs like profilin, ciboulot, Tβ4, twinfilin, and DBP prevent spontaneous nucleation. With these 1∶1 complexes, some (profilin and ciboulot) still allow addition at the barbed end of actin filaments, and others (Tβ4, twinfilin, and DBP) just sequester G-actin. Gelsolin servers and then caps the barbed end of actin filaments.
Figure 2.
Structures of seven ABPs complexed with G-actin.
(A) G-actin, with subdomains shown in magenta, green, yellow, and red, respectively. (B) Profilin. (C) Twinfilin ADF-homology domain 2. (D) Gelsolin domain 1. (E) WASP WH2, linker, and central segments. (F) Ciboulot domain 1. (G) Thymosin β4. (H) Vitamin-D binding protein. The cleft-lying helices of all seven ABPs are shown in yellow. The C-terminal portions of the ABPs in (E) and (F), shown in light color, are modeled and can easily dissociate. In (H) the three domains of DBP are in dark blue, cyan, and light blue respectively.
Figure 3.
Three classes of association mechanisms.
(A) A relatively rigid globular ABP like profilin reaches the transient complex (not shown) with G-actin by diffusion and then forms the contacts nearly all at once to produce the native complex. The binding of twinfilin ADF-homology domain 2 and gelsolin domain 1 to G-actin follows the same mechanism. (B) The dock-and-coalesce mechanism for the G-actin binding of WASP WCA, an intrinsically disordered protein. (C) Vitamin-D binding protein has an opening between its domains 1 and 3, in both the free state and the G-actin-bound state, that is too narrow for G-actin to enter. So DBP must undergo breathing motion to transiently widen its opening. Once G-actin is inside, the opening quickly narrows to clamp around G-actin. In (A) G-actin has the orientation as in Figure 1; in (B) it is rotated clockwise (viewed from top) around a vertical axis by 45°; and in (C) it is rotated counterclockwise (viewed from top) around a vertical axis by 90°.
Table 1.
Experimental and calculated rate constants for the association of seven actin-binding proteins with G-actin.
Table 2.
Geometric and electrostatic properties of seven actin-binding proteins with G-actin.
Figure 4.
Identifying the docking segments for WASP WCA, ciboulot domain 1, and Tβ4 in their binding to G-actin, by finding the fragments with the highest rate constants for the docking step.
The sequences of the three proteins are aligned. Regions forming helices are indicated by cylinders. In all three cases, the proposed docking segments are the fragments ending at the third position of the conserved “LKK” motif (ending residues shown in red). The corresponding rate constants for the docking step are indicated by a vertical dashed line. Inclusion of the C-terminal residues in italic in the proposed docking segments produced large gaps in Nc, indicating that these residues must belong to the coalescing segments instead.