Molecular architecture of the Spire–actin nucleus and its implication for actin filament assembly

Abstract

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs-even a single WH2 repeat-sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.cytoskeleton | nucleation T he first step in the assembly of actin filaments is nucleation (1, 2). Three major classes of nucleating proteins have been identified until today: the Arp2/3 complex together with newly identified nucleation-promoting factors such as WASH, WHAMM and JMY (3-7), formins (8, 9), and a third class which comprises the proteins commonly named tandem-monomer-binding nucleators (10). This last group of nucleators contains 17-27 amino acid long actin-binding motifs called the WH2 repeats-the name derived from the Wiskott-Aldrich syndrome protein homology domain 2 (11, 12). The group consists of Spire (13), , Leiomodin from muscle cells (15), JMY (6), and the recently discovered adenomatous polyposis coli (APC) protein (16). These proteins share the common ability, mediated by WH2 domains, to gather actin monomers into a nucleation complex, but the arrangement of nuclei might vary significantly among these nucleators. Spire contains four consecutive WH2 domains and is the most important representative member of the group. The molecular mechanism of actin nucleation is well described for the Arp2/3 complex (17) and formins (18), whereas several different mechanisms have been proposed for Spire (19)(20)(21)(22), Leiomodin (15), JMY (6), and the APC protein (16). The N-terminal domain of Spire (SpireNT, residues 1-520 in Drosophila melanogaster Spire; see Fig. S1) has the potential to form a string of four actin monomers through the interaction with four WH2 repeats (13,19,20). WH2 motifs are known to be intrinsically disordered, adopting an α-helical structure only upon binding to actin (23). Alignments of WH2 domains indicate that the most conserved regions are the LKK motif and an α-helix at the N terminus, which is shown to be the principal structural actin-binding element that binds to actin in its hydrophobic pocket between actin's subdomains 1 and 3 (20,24,25). The rest of the WH2, including the LKK motif, exten...

Structural Basis of Actin Filament Nucleation by Tandem W Domains

“…Therefore, the non-filament-like conformation of actin-Cobl-2W nucleus revealed in our structure is biologically relevant. In fact, crosslinking and small-angle X-ray scattering studies of tandem W domains in Spire concluded that the actin-Spire-4W complex is also not strictly filament like (Chen et al, 2012; Ducka et al, 2010; Sitar et al, 2011). …”

Multiple Forms of Spire-Actin Complexes and their Functional Consequences

“…The absence of strong longitudinal actin cross-linking in the presence of Spir-ABCD suggests that the contacts are not precisely filament-like. Sitar et al (27) consistently predict elongated structures for Dm Spir bound to actin from small angle x-ray scattering data. They also conclude that these complexes are not strictly filament-like.…”

“…Furthermore, earlier EM studies showed little difference between Spir-NT and Spir-ABCD in complex with actin (lengths of 22 Ϯ 4 versus 26 Ϯ 5 nm, respectively; (1)). Likewise, rigid body modeling of small angle x-ray scattering data by Sitar et al (27) produced little difference between the WH2 domains and actin of Spir-NT and Spir-ABCD.…”

Drosophila Homologues of Adenomatous Polyposis Coli (APC) and the Formin Diaphanous Collaborate by a Conserved Mechanism to Stimulate Actin Filament Assembly

“…It is now clear that formins utilize a variety of domains and partners to accomplish nucleation, including the monomer binding DAD domain (5,41) and direct collaboration with actin monomer-binding NPFs including Bud6, APC, and Spire (9,10,23,(42)(43)(44)(45)(46)(47)(48). These NPF activities can be mediated by a variety of domains, including WASp homology 2 (WH2) or WH2-like domains (WASp, Bud6, and Spire), the Basic domain (APC), and Gelsolin repeats (Fli-I) (7,9,10,23,(42)(43)(44)(45)(46)(47)(48). Despite these structural differences, a number of common mechanistic features are shared among most of these NPFs, such as binding to the formin C-terminal tail, binding to actin monomers, and interacting with microtubules.…”