Structural insights into de novo actin polymerization

Abstract

Many cellular functions depend on rapid and localized actin polymerization/depolymerization. Yet, the de novo polymerization of actin in cells is kinetically unfavorable because of the instability of polymerization intermediates (small actin oligomers) and the actions of actin monomer binding proteins. Cells use filament nucleation and elongation factors to initiate and sustain polymerization. Structural biology is beginning to shed light on the diverse mechanisms by which these unrelated proteins initiate polymerization, undergo regulation, and mediate the transition of monomeric actin onto actin filaments. A prominent role is played by the W domain, which in some of these proteins occurs in tandem repeats that recruit multiple actin subunits. Pro-rich regions are also abundant and mediate the binding of profilin-actin complexes, which are the main source of polymerization competent actin in cells. Filament nucleation and elongation factors frequently interact with Rho-family GTPases, which relay signals from membrane receptors to regulate actin cytoskeleton remodeling.

Figure 1

Domain diagram of DRFs and representative structures. (a) A prototypical DRF (domain boundaries correspond to mDia1) consists of the following domains: GBD (GTPase-binding domain), DID (diaphanous inhibitory domain), DD (dimerization domain), CC (coiled coil), FH1 and FH2 (formin homology 1 and 2 domains) and DAD (diaphanous autoregulatory domain). (b-c) Structures of N-terminal regulatory fragments with bound RhoC [9] and DAD [10]. Ribbon diagrams colored according to the domain diagram in part a. (d) Illustrations of the structure of the FH2-actin complex [12] showing, in a sequence, the crystallographic FH2 dimer (obtained after domain swapping the ‘lasso’ region from the lower subunit), the actin subunits bound to the dimer (blue and cyan), and a third actin from the complex below (gray). Each FH2 domain is structurally subdivide into lasso, linker, knob, coiled coil and post regions. This figure is inspired by similar figures in [3].

Figure 2

Structure of the W-actin complex. The structure shown [15] is that of the W domain of the NPF protein WAVE2 (red) bound to actin (blue). Subdomains 1-4 of actin are labeled. The N-terminal helix of the W domain binds in the hydrophobic or target-binding cleft of actin, a common binding site for actin-binding proteins [14]. In WAVE2 the conserved LKKT(V) motif correspond to ⁴⁴⁹LRRV⁴⁵² (side chains shown).

Figure 3

Tandem W domain-based filament nucleators. Domain boundaries defined according to the sequences of Drosophila Spire (Q9U1K1), mouse Cobl (Q5NBX1) and Vibrio parahaemolyticus VopL (Q87GE5). Cartoons above the tandem W repeats depict the proposed nucleation mechanisms of Spire [16] and Cobl [17]. VopL’s nucleation mechanism is still unknown. Pro-rich sequences and potential dimerization domains are colored magenta and green, respectively.

Figure 4

Actin filament nucleation by NPFs-Arp2/3 complex and NPFs. (a) Domain boundaries of three representative NPF proteins: mouse JMY (Q9QXM1), mouse N-WASP (Q91YD9) and human WAVE2 (Q9Y6W5). Note the resemblance with the filament nucleators shown in Figures 3, including the presence of Pro-rich regions and varying numbers of W domains (1 to 3). (b) The Arp2/3 complex consists of seven proteins, including the actin related proteins Arp2 and Arp3 and subunits ARPC1 to 5 (labeled 1 to 5). The WCA region of NPFs (contoured in part a) is the smallest fragment capable of catalyzing the formation of a polymerization nucleus, consisting of the two Arps and one to three actin subunits, and a conformational change in Arp2/3 complex that promotes monomer addition to the branch filament and binding of the nucleus to the side of a preexisting filament (mother filament). The branch grows from the barbed ends of the Arps at a 70° angle with respect to the mother filament. (c) Structure of inactive Arp2/3 complex [36,37]. Subdomains 1 and 2 of Arp2 are disordered in the structures, but were added here by analogy with actin. The Arp2/3 complex subunits are colored according to the diagram of part b. (d) SAXS-derived model of WCA-actin-Arp2/3 complex [43]. The orientation is the same as in part c. The mother and branch filaments are shown for reference, although this work did not address branch assembly. Note that Arp2 is proposed to move up compared to its location in part c. This study placed the first actin subunit of the branch at the barbed end of Arp2. The position of the W domain is known from the crystal structure of its complex with actin (ses Figure 2) and imposes constraints on the location of the C motif, which may bind in the hydrophobic cleft of Arp2. The position of the A motif (pink) is less well constrained, but this model is consisting with it binding at the interface between Arp3 and ARPC3, as suggested by biochemical studies [39-42].

Figure 5

Domain diagram of VASP and representative structures. (a) Domain boundaries correspond to human VASP (P50552). Notice the resemblance with N-WASP (Figure 4a). (b) Structure of the EVH1 domain of Mena in complex with a Listeria ActA peptide [57]. (c) Structure of the ternary complex of profilin-actin with a VASP fragment comprising the last Pro-rich profilin-binding site and GAB domain [46]. The Gly-rich linker between these two domains was not visualized (discontinuous line). (d) Structure of the tetramerization domain, revealing a right-handed coiled-coil fold [59].