Structure and activity of full‐length formin mDia1

Abstract

Formins are a conserved family of actin assembly-promoting factors with essential and diverse biological roles. Most of our biochemical understanding of formin effects on actin dynamics is derived from studies using formin fragments. In addition, all structural information on formins has been limited to fragments. This has left open key questions about the structure, activity and regulation of intact formin proteins. Here, we isolated full-length mouse mDia1 (mDia1-FL) and found that it forms tightly autoinhibited dimers that can only be partially activated by RhoA. We solved the structure of autoinhibited mDia1-FL using electron microscopy and single particle analysis. Docking of crystal structures into the 3D reconstruction revealed that the fork-shaped N-terminal DID-CC region hangs over the ring-shaped FH2 domain, suggesting that autoinhibition results from steric obstruction of actin binding. Deletion of the C-terminal DAD domain extended mDia1 structure and activated it for actin assembly. Using TIRF microscopy, we observed that RhoA-activated mDia1-FL persistently accelerated filament elongation in the presence of profilin similar to mDia1 FH1-FH2 fragment. These observations validate the known activities of FH1-FH2 fragments as reflecting those of the intact molecule. Our results further suggest that mDia1-FL does not readily snap back into the autoinhibited conformation and dissociate from growing filament ends, and thus additional factors may be required to displace formins and restrict filament length.

Initiation of lamellipodia and ruffles involves cooperation between mDia1 and the Arp2/3 complex

“…S3I, Movie 4). Taken together, these data indicate that full-length mDia1 is an autoinhibited actin nucleator that controls also actin filament elongation, as previously suggested by mDia1 deletion mutants (Kovar et al, 2006;Romero et al, 2004) and quasi full-length mDia1 (Maiti et al, 2012). Next, we combined mDia1 with WAVE2 ( Fig.…”

Actin Monomers Activate Inverted Formin 2 by Competing with Its Autoinhibitory Interaction

“…It is unclear whether this mechanism is generally applicable, although actin monomer binding to regions surrounding the DAD occurs for several formins (13)(14)(15). mDia1 appears to be autoinhibited even in the presence of free actin monomers (4,32), which might be due to the extremely low affinity of its DAD region for actin or to the fact that the actin binding site does not overlap the DID binding site (13, 14). The C-terminal region of FMNL3 binds actin monomers with micromolar affinity, but the region responsible for this binding is distinct from the canonical DAD region.…”

Formins at a glance

“…DRFs are autoinhibited through DID-DAD interactions. Recent crystallographic and single-particle electron microscopy studies show that, in the autoinhibited conformation, the N-terminus physically obstructs the ability of the C-terminus to polymerize actin (Nezami et al, 2010;Otomo et al, 2010;Maiti et al, 2012). Binding of active Rho-GTPases to GBD activates the formin by releasing these DID-DAD interactions.…”

“…Binding of active Rho-GTPases to GBD activates the formin by releasing these DID-DAD interactions. However, activation is noticeably incomplete (Li and Higgs, 2003;Maiti et al, 2012), suggesting that other factors are required to fully activate formins.…”