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

Abstract

Three classes of proteins are known to nucleate new filaments: the Arp2/3 complex, formins, and the third group of proteins that contain ca. 25 amino acid long actin-binding Wiskott-Aldrich syndrome protein homology 2 domains, called the WH2 repeats. Crystal structures of the complexes between the actin-binding WH2 repeats of the Spire protein and actin were determined for the Spire single WH2 domain D, the double (SpirCD), triple (SpirBCD), quadruple (SpirABCD) domains, and an artificial Spire WH2 construct comprising three identical D repeats (SpirDDD). SpirCD represents the minimal functional core of Spire that can nucleate actin filaments. Packing in the crystals of the actin complexes with SpirCD, SpirBCD, SpirABCD, and SpirDDD shows the presence of two types of assemblies, "side-to-side" and "straight-longitudinal," which can serve as actin filament nuclei. The principal feature of these structures is their loose, open conformations, in which the sides of actins that normally constitute the inner interface core of a filament are flipped inside out. These Spire structures are distant from those seen in the filamentous nuclei of Arp2/3, formins, and in the F-actin filament.cytoskeleton | X-ray crystallography | fluorescence assay T he actin cytoskeleton is involved in many cellular processes, including cell motility, cell adhesion, endo/exocytosis, intracellular and membrane trafficking, and the maintenance of cell shape and polarity. These cellular functions require the dynamic remodeling of the actin cytoskeleton, which depends on the transition between monomeric actin (G-actin) and its filamentous state (F-actin) (1). The initiation of actin polymerization from free actin monomers requires nucleation factors that help to overcome the kinetic barrier for formation of actin dimers and trimers (2, 3). Three classes of actin-nucleation proteins have been identified until today: the Arp2/3 complex together with newly recognized nucleation-promoting factors such as WASH, WHAMM, and JMY (4-8), formins (9-12), and the third group of proteins that contain 17-27 amino acid long actin-binding motifs called the WH2 repeats-the name derived from the WASP (Wiskott-Aldrich syndrome protein) homology domain 2 (13-15). The third group consists of Spire (16), Cordon-bleu (17), and Leiomodin from muscle cells (18). The molecular mechanism of actin nucleation is well described for the Arp2/3 complex (19,20) and formins (21), whereas little molecular details are known about the nucleation by Spire.Spire was first identified as an actin-binding factor necessary for the correct establishment of polarity axes of the oocyte in Drosophila (22). It mediated actin-microtubule interactions and has important roles in membrane transport, although the upstream signaling pathways that regulate these functions have not been fully studied (23)(24)(25). Spire is a 1,020 amino acid long, multidomain protein (Fig. 1A); the most important domains, from the point of view of actin organization, are localized in the N-terminal part of the protein...

Ablation of cyclase-associated protein 2 (CAP2) leads to cardiomyopathy

“…Actin purification, labeling, and fluorescence measurements were carried out using procedures described previously [10].…”

“…The WH2 domain is a small motif of approximately 25 amino acids found in different proteins such as WASP (Wiskott-Aldrich Syndrome Protein), thymosin, Spire, Cordon-bleu, Leiomodin, and JMY. These proteins can either sequester monomeric actin or inhibit actin polymerization like thymosin or nucleate actin assembly like Spire, Cordon-bleu, and JMY [10]. WH2 motifs are intrinsically disordered, adopting an a-helical structure only upon binding to actin [11].…”

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

“…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, extends along the outer surface of the actin subdomain 1 up to subdomains 2 and 4.…”

“…However, recent high-resolution crystallographic studies of a number of Spire-actin constructs composed of one to four WH2 domains including the linkers did not support this view and showed arrangements that significantly differed from the structure of actin filaments (20). The 7-Å resolution crystal structure of an (actin)2-(WH2)3 complex (21) was also interpreted as different from the long-pitch helix of the actin filament; however, our recalculations using the deposited intensity and model data (21) did not verify, by the usual crystallographic reliability criteria, the suggested solution of the crystal structure, which we therefore regard as insignificant.The smallest of these complexes, actin/single-WH2 repeat (actin-ðWH2Þ 1 ), displayed the bound α-helix and the LKK motif well defined at atomic resolution (20). All other complexes with more than one WH2 domain were isomorphous and showed precisely the polypeptide main chain of the α-helical actin-binding element, but blurred electron density for the amino acid side chains, as expected for a statistical averaging of the homologous sequences of the WH2 domains.…”

“…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).…”