Our data demonstrate that multiple WH2 motifs and the VCD of VopL are both required for high potency actin filament nucleation. Dimerization, either by the VCD or artificially by GST, is necessary for the WH2 motifs to nucleate. The VCD itself also appears to contact actin, since it alone can nucleate weakly, and mutagenesis of conserved residues in the base and arm decreases activity. These key data, along with the geometry of the VCD structure, suggest a preliminary model for the actin nucleus assembled by VopL. A central concept in this model is that the WH2 motifs recruit actin monomers, while the VCD provides organization that leads to a productive nucleus.
The requirement for dimerization of the WH2 element suggests that in the nucleus, at least one actin monomer is contributed or recruited by each subunit. An individual multi-WH2 protein should be able to assemble a single strand of the nascent paired actin filament (i.e. stabilize long-pitch contacts between actin monomers). But such activity is evidently insufficient for nucleation by VopL. This observation suggests that in VopL dimers, the paired WH2 elements act by stabilizing lateral (short-pitch) actin-actin contacts. Thus, the nucleus should minimally contain a short-pitch actin dimer.
How might such a structure interact with the VCD? One possibility is suggested by geometric considerations. In a short pitch actin dimer, the two subunits are axially displaced on average by 27.5 Å39
. In the VCD, the distal end of the arm is ~30 Å from the platform created by the base. Thus, the axial displacement of an actin dimer could be accommodated by only small changes in the structural organization of the VCD. In this configuration the penultimate actin monomer would contact the distal end of the arm and the terminal monomer would contact the base (). While our image in has the plane of the terminal monomer perpendicular to the plane of the VCD, other orientations with more acute angles are also possible. Such an arrangement would be consistent with several observations. First, the VCD alone has nucleation activity, suggesting that it may contact both actins in a short pitch dimer. Our mutagenesis data () suggest that residues at both the distal end of the arm and the VCD base formed by base contribute to nucleation activity, probably through contacts to actin. Second, in order for WH2c to bind this penultimate actin, the WH2c-VCD linker would need to extend from the N-terminus of the VCD, around the arm to its tip, a distance of ~50 Å, and then approximately half the length of an actin monomer to the N-terminus of the WH2 motif35
, ~28 Å. While these distances are obviously very approximate, they would be consistent with the idea that the natural 24 residue linker (~80 Å fully extended) may be poorly able to position a WH2c-bound actin on the VCD, explaining the low activity of W1
-C, and the progressive increase in activity as the linker is lengthened (). In native VopL, the penultimate monomer may be recruited by WH2b. Third, SAXS analyses from Dominguez and colleagues on the W1
-C:actin complex suggest that an ordered actin contacts the base 37
. This terminal monomer could be recruited by the WH2c of the opposing VCD. Finally, the short-pitch dimer bound to the VCD would be further stabilized by additional actin monomers recruited by additional WH2 motifs, consistent with the activity series W3
-C > W2
-C > W1
-L-C (). While this model remains speculative, it makes testable predictions that will be explored in future work.
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 (more ...)
The stabilization of short-pitch contacts in the nascent actin filament appears to be a common feature of WH2-based nucleation factors, both prokaryotic and eukaryotic. In Cobl and Lmod, the stoichiometry of actin binding and linker length dependencies of activity have led to models in which WH2 and other motifs stabilize a short-pitch actin trimer8,9
. In Spire, a tandem array of WH2 motifs was shown by electron microscopy to organize a linear actin structure, which was proposed to nucleate through serving as a single, long-pitch strand of an actin filament7
. However, Spire alone is a relatively weak nucleation factor, whose activity is appreciably enhanced through dimerization mediated by the formin protein, Cappuccino40
. Similarly, the actin nucleation factor TARP from the pathogen Chlamydia trachomatis
requires oligomerization of its WH2 motif by an adjacent poly-proline region for activity33
. In both Spire and TARP, higher potency likely arises, as in VopL, from the ability of dimers to organize lateral contacts between actin monomers in the nascent filament. These experimental findings are all consistent with computational analyses of actin nucleation, which suggest that a stabilized short-pitch dimer will recruit a third monomer with much greater affinity than will a long-pitch dimer4
. Thus, short-pitch dimers should act as more effective nuclei.
Together, our findings provide an initial mechanistic model for the potent actin nucleation activity of VopL. Yet many questions remain to be answered. What is the conformation of the VopL-bound actin nucleus, and what contacts to both WH2 motifs and the VCD stabilize it? Does VopL remain bound to filaments after nucleation? If so, does it bind filament ends or sides; does it remain static like the Arp2/3 complex or process like formins? If not, what triggers its release from the nascent filament? Why do VopL and VopF, which presumably nucleate through very similar mechanisms, produce different actin structures in cells? Our work here provides an initial framework to address these questions and others in the future.