Journal of Molecular Biology
Structure of the FH2 Domain of Daam1: Implications for Formin Regulation of Actin Assembly
Introduction
Formins are a large family of proteins that regulate actin filament assembly in response to diverse signals, and they are crucial for a variety of actin-dependent cellular processes including polarized cell growth, vesicular transport and cytokinesis.1., 2., 3., 4. Characteristic features of this protein family include two commonly shared domains, the formin-homology-1 (FH1) and formin-homology-2 (FH2) domains.5 The FH1 domain, formed by proline-rich stretches, binds profilin as well as SH3 domain-containing proteins. The FH2 domain binds actin, and directly nucleates new unbranched actin filaments.6,7 Additionally, the FH2 domain has been shown to remain associated with the barbed end of the actin filament as it elongates.8., 9., 10., 11., 12., 13.
A recent phylogenetic analysis revealed that metazoan FH2 domains segregate into seven subfamilies; three of these groups, Dia(diaphanous), Daam(dishevelled-associated activator of morphogenesis) and FRL(formin-related gene in leukocytes) possess some similarities outside of the FH2 domain,14 and have been termed “diaphanous-related” formins. They share a common domain structure that includes a GTPase-binding domain (GBD), a diaphanous inhibitory domain (DID), and a coiled-coil region followed by the FH1, FH2 and C-terminal diaphanous auto-regulatory domain (DAD)2,15 (Figure 1(a)). These formins are thought to be auto-inhibited via intramolecular binding of the N-terminal DID to the C-terminal DAD.16., 17., 18., 19., 20. Binding of GTP-bound Rho GTPases to the GBD domain and adjacent DID domain displaces the auto-inhibitory interaction, at least partially releasing the FH2 domain to activate actin assembly.17,19., 20., 21., 22.
Daam1 was first studied as a novel formin protein involved in the planar cell polarity pathway in Xenopus gastrulation.23 Habas et al. demonstrated that Daam1 is required for Wnt/Fz signaling in Xenopus gastrulation via direct interactions with both dishevelled and RhoA.23 The “DEP” and “PDZ” domains of dishevelled were demonstrated to bind a C-terminal fragment of Daam1 that spans its FH1, FH2, and DAD domains (residues 490–1078), while RhoA was shown to bind the N-terminal region as in other diaphanous-related formins. In Drosophila, Daam is required for organizing the apical actin cables that define the taenidial fold pattern of the tracheal cuticle, but does not appear to be required for planar cell polarity signaling.24 As with other diaphanous-related formins, Daam1 binds to RhoA and Cdc42 in a GTP-dependent manner.25
The formin FH2 domain is the minimal functional unit for the nucleation and elongation of actin filaments.7,8,26 The crystal structure of the FH2 domain of budding yeast Bni1 revealed the overall architecture of the domain and showed that it forms a stable and flexible ring-shaped dimer.27 Analysis of the truncated, monomeric form of the mDia1 FH2 domain of mDia1 revealed a similar rod-shaped core.28 Functional analysis of conserved surface residues in these studies identified two actin binding surfaces, one at either end of each rod-shaped subunit.27 Co-crystallization of the Bni1 FH2 domain with tetramethyl-rhodamine-labeled actin showed that these conserved patches interact with distinct actin subunits, allowing each of half the FH2 dimer to “bridge” between two or three actin subunits.29 Together with functional analysis of formin-mediated actin assembly, these studies suggest a general model of FH2 domain function in which the FH2 dimer can nucleate a new filament by stabilizing an actin dimer (or possibly a higher order oligomer), and processively move with and cap the elongating barbed end via a “stair-stepping” motion made possible by the flexible tethered-dimer construction of the domain.9,11,13,27,30 Essentially, one subunit can move to adopt an “open” configuration to accept a new actin subunit, while the other remains associated in a “closed” configuration.31 The adjacent FH1 domain accelerates filament elongation by interacting with profilin–actin complexes and shuttling actin to the barbed end.8., 9., 10.,31
Although the overall structure and general mechanism of the FH2 domain are likely conserved, the rates of nucleation and assembly and profilin dependence of FH2 domains vary greatly.1,9 As there are at least 15 distinct formins in mammals,2 it is important to understand their respective structural similarities and differences. Here we describe the structure of a fragment of human Daam1 (residues 596–1078) containing the FH2 domain. The Daam1 FH2 domain consists of an N-terminal “lasso” segment, a flexible linker that is largely disordered in the present structure, and a rod-shaped domain formed by three sub-domains termed the “knob”, “coiled-coil”, and “post”. As in the Bni1 FH2 domain, the Daam1 domain forms a head-to-tail dimer stabilized by the N-terminal “lasso” segment in each subunit, which wraps around the post in the other subunit in the dimer. Although the overall “tethered dimer” architecture is quite similar to that of the yeast Bni1 FH2 domain, we find differences that may be functionally important. In particular, divergence in the lasso/post dimerization interface may prevent heterodimerization of Daam1 with other formins. Additionally, the two halves of the Daam1 dimer pack together in a manner that occludes their actin binding surfaces and is suggestive of an inactive conformation of the FH2 domain. Our structure/function studies of the Daam1 FH2 domain show that indeed the wild-type domain has only very weak actin assembly activity as compared with other mammalian formins (mDia1 and mDia2), but mutations that disrupt the putative auto-inhibitory interactions increase actin assembly about tenfold. These “derepressed” mutants have activity similar to other formins studied to date.
Section snippets
Overall structure
The structure reveals the tethered-dimer architecture predicted to be characteristic of all FH2 domains.27 The Daam1 FH2 domain is composed of five sub-domains: the lasso, a flexible linker, a globular knob sub-domain, a coiled-coil region and a post sub-domain with a C-terminal helical extension (Figure 1(b)). In the dimeric molecule, the two subunits are arranged in a head-to-tail fashion and the lasso of each subunit encircles the post sub-domain of the other (Figure 1(c)). In the present
Concluding Remarks
The structure described here provides only the second view of a complete, dimeric formin FH2 domain, and it reveals a locked, inactive conformation stabilized by β-strand interactions between the hemidimers. Sequence and structural comparisons of the Daam1 FH2 structure with Bni1 and other FH2 domains also show that the overall structure of the lasso/post dimerization motif is preserved, but that key differences in dimer contacts may facilitate homodimerization over heterodimerization.
Protein expression and purification
A DNA fragment encoding amino acids 596–1078 of human Daam1 was amplified by PCR and sub-cloned into a modified pET vector containing an amino-terminal His6 tag with a tobacco etch virus (TEV) cleavage site. The S612R, A651P, Q653P and I698A mutations were introduced into this construct using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, California, United States) according to the manufacturer's protocol. All of the constructs were confirmed by DNA sequencing. The
Acknowledgements
We thank John Wallingford and Xi He for providing the Daam1 cDNA and Azin Nezami for helpful discussions. This work was supported in part by a NIH grant GM071834 (to M.J.E.). M.J.E. is the recipient of a Scholar Award from the Leukemia and Lymphoma Society.
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