Structure and function of the interacting domains of Spire and Fmn-family formins

Abstract

Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.M any processes in the eukaryotic cell depend upon the timely generation and disassembly of actin filaments. The ratelimiting step of filament formation is the creation of a stable actin nucleus. At least three different classes of proteins have evolved to accelerate this step: formins, the Arp2/3 complex, and WiscottAldrich homology 2 (WH2)-domain nucleators (1). How actin nucleators from different classes cooperate to build particular actin structures is an area of intense interest and investigation. For example, the direct biochemical and genetic links between WH2-based Spir (2, 3) and the formin Cappuccino (4) suggest close mechanistic collaboration between these proteins in actin assembly (5-7).Spire (spir) and cappuccino (capu) were first identified in screens for genes affecting embryonic pattern formation in Drosophila melanogaster (Dm) (7). They synergize to build a cytoplasmic actin mesh, and mutation of either of these genes results in loss of this structure, premature microtubule-dependent cytoplasmic streaming, and gross defects in embryonic morphology (8, 9). Mice lacking formin-2 (Fmn2; a mammalian Capu ortholog) exhibit egg failure and female hypofertility (10) due to loss of an actin-based structure during meiosis (11,12), supporting the functional conservation of these proteins in higher eukaryotes.Formins possess an actin-nucleating formin homology 2 (FH2) domain and an adjacent proline-rich FH1 domain (Fig. 1A). The FH2 domain remains bound to the barbed end of the actin filament as additional subunits are added, protecting growing ends from the activity of capping proteins, which would otherwise terminate elongation (13). Fmn-family formins, including Capu and orthologs Fmn1 and Fmn2 (5,14,15), lack obvious regulatory domains found in diaphanous-related formins (DRFs), which include a GTPase-binding domain, diaphanous inhibitory domain, and C-terminal diaphanous autoregulatory domain (DAD) (16,17). Fmn-family formins do have short (approximately 25 ...

Baculovirus protein PK2 subverts eIF2α kinase function by mimicry of its kinase domain C-lobe

“…The protein interaction function of KIND domains has been proposed to derive from a vestigial remnant of substrate recognition infrastructure. However, the crystal structure of the KIND domain of Spir in complex with interaction peptides did not resemble the substrate-targeting mode for any kinase solved to date and instead may have evolved independently via deletion of a conserved structural element of the kinase C-lobe (28,29). Our study raises the intriguing possibility that, in addition to binding peptides, the KIND domain also may bind to protein kinase domains to regulate their catalytic function.…”

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

“…The linkers and extensions were modeled as flexible dummy amino acid chains, which allowed free actin-WH2 domain movement within the limits set by the linker lengths in the protein. For SpireNT/actin, the recently released KIND domain structures (PDB codes 2YLF and 3RBW) (32,33) were used as an additional rigid body element. The models obtained by rigid body refinement are in agreement with the fitting of the crystal-derived models, indicating the longitudinal-like arrangement being the only one or at least the dominant conformation of the Spire-actin complex.…”

“…Taking into account our solution structure of SpireNT/actin complex and the recently released crystal structures of the Spire KIND domain complexed with the C-terminal region of Fmn2 formin (32,33), we propose the model shown in Fig. 5 E-H, in which one formin dimer binds two molecules of Spire, and each Spire binds to four actin molecules, bringing together eight actin molecules, which then could form a complete filament seed and thereby create a nucleating complex.…”

Membrane Targeting of the Spir·Formin Actin Nucleator Complex Requires a Sequential Handshake of Polar Interactions

“…The introduction of a positive charge into the acidic groove of the KIND domain (human Spir-1 Y134K and Drosophila Spir Y232K mutations) strongly impaired the Spir/formin interaction in vitro (18,19). Transgenic expression of Drosophila Spir Y232K mutant protein in flies having a spir mutant background failed to rescue the oocytic actin meshwork (14).…”

“…The KIND domain evolved from the catalytic protein kinase fold into a protein interaction module (10, 16 -19). The complex crystal structure disclosed a large interface of positively charged residues of the formin-2 FSI sequence mediating contacts to an acidic groove at the surface of the Spir-1-KIND domain (18,19).…”