X-ray scattering study of actin polymerization nuclei assembled by tandem W domains

Abstract

The initiation of actin polymerization in cells requires actin filament nucleators. With the exception of formins, known filament nucleators use the Wiskott-Aldrich syndrome protein (WASP) homology 2 (WH2 or W) domain for interaction with actin. A common architecture, found in Spire, Cobl, VopL, and VopF, consists of tandem W domains that tie together three to four actin monomers to form a polymerization nucleus. Uncontrollable polymerization has prevented the structural investigation of such nuclei. We have engineered stable nuclei consisting of an actin dimer and a trimer stabilized by tandem W domain hybrid constructs and studied their structures in solution by x-ray scattering. We show that Spire-like tandem W domains stabilize a polymerization nucleus by lining up actin subunits along the long-pitch helix of the actin filament. Intersubunit contacts in the polymerization nucleus, thought to involve the DNase I-binding loop of actin, coexist with the binding of the W domain in the cleft between actin subdomains 1 and 3. The successful stabilization of filament-like multiactin assemblies opens the way to the crystallographic investigation of intersubunit contacts in the actin filament.actin nucleation ͉ cytoskeleton dynamics ͉ WH2 domain T he spontaneous polymerization of actin filaments is inhibited in cells by actin monomer-binding proteins such as profilin and thymosin-␤4 (T␤4). Cells use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting during actin assembly (1). In addition to Arp2/3 complex and formins (2), a number of filament nucleators have been recently discovered: Spire (3), Cobl (4), VopL (5), VopF (6), and Lmod (7). With the exception of formins, all filament nucleators use the W domain for interaction with actin.The W domain is a short actin-binding motif of 17-27 aa (8). The N-terminal portion of the W domain forms a helix that binds in the cleft between actin subdomains 1 and 3 at the barbed end of the actin monomer (9). After this helix, the W domain presents an extended region that climbs toward the pointed end of the actin monomer. This portion of the W domain has variable length and sequence but comprises the conserved four-aa motif LKKT(V). T␤4, a member of the W domain family, contains an additional helix at the C terminus, which binds atop actin subdomains 2 and 4 and caps the pointed end (10).The W domain frequently occurs in tandem repeats. Tandem W domains constitute a common architecture among actin filament nucleators, such as Spire, Cobl, VopL, and VopF (Fig. 1A). The actin monomers bound to the individual W domains of these proteins are thought to come together to form a filamentlike nucleus for polymer assembly. This proposal, however, has not been directly demonstrated. An apparent contradiction is that the cleft between actin subdomains 1 and 3, where the N-terminal helix of the W domain binds, is also thought to participate in intersubunit contacts in the actin filament (11-13). Therefore, it remains to be demonstrated whether and...

Structures of actin-bound Wiskott-Aldrich syndrome protein homology 2 (WH2) domains of Spire and the implication for filament nucleation

“…There was, however, little experimental evidence to support such a model (which has different packing of actin molecules from that of our straight-longitudinal configuration). Recent X-ray scattering analysis of the complex of actin with an artificial tandem of WH2 domains could be interpreted as if a Spire-like protein promotes polymerization by aligning actin subunits in a single strand along the long-pitch helix of the actin filament (34). We, however, propose that the model described by Rebowski et al (34), which would also be compatible with our data, shows a final stage of the nucleation rather than the initial step of the actin seed formation.…”

The WH2 Domain and Actin Nucleation: Necessary but Insufficient

“…Importantly, linker-3 (between WH2 domains 3 and 4) was found to play a crucial role in Spire nucleation. Rotary-shadowed electron microscopy [6] and small angle x-ray scattering [11] suggest that when the linkers between WH2 domains are short as in Spire (13 to 15 amino acids), such repeats stabilize linear arrays of actin subunits along the long-pitch, two-start filament helix (Figure S1). However, this arrangement appears suboptimal for nucleation, presumably because the long-pitch helix of the double-stranded filament is not an ideal catalyzer of nucleation [13], and the flexible linkers between WH2 domains cannot properly align the actin subunits for nucleation.…”

“…As all the structures of WH2-actin complexes invariably show, the WH2 domain consists of 17-20 amino acids ( Figure 2 ), folded as an N-terminal α-helix, which binds in the mostly hydrophobic target-binding cleft at the barbed end of the actin monomer, and an extended C-terminal portion that raises along the actin surface, roughly following the ridge between the outer (subdomains 1 and 2) and inner (subdomains 3 and 4) domains of the actin monomer [5, 68]. This binding site overlaps only partially with inter-subunits contacts in the filament [11, 12, 69] (see also Online Supplemental Information Figure S1), and makes binding of the WH2 domain sensitive to the nucleotide state of the actin monomer, showing a clear preference for polymerization-ready ATP-bound monomers [68, 70-72]. The extended C-terminal region comprises the so-called LKKT motif, a nomenclature resulting from the existing relationship between the WH2 domain and thymosin-β4 [68, 73].…”

Structural Basis of Actin Filament Nucleation by Tandem W Domains

“…Tandem W domains are expected to “stitch” together multiple actin subunits to overcome the largest energy barriers in actin nucleation, the formation of actin dimers and trimers, thus accelerating actin polymerization. Despite various efforts in the past (Rebowski et al, 2008, 2010), this process remains poorly understood, owing to the inability to obtain high-resolution structures of native multisubunit actin nucleus, which, once formed, rapidly proceeds to long and dynamic actin filament.…”