Abstract

Formins induce the nucleation and polymerisation of unbranched actin filaments via the formin-homology domains 1 and 2. Diaphanous-related formins (Drfs) are regulated by a RhoGTPase-binding domain situated in the amino-terminal (N-terminal) region and a carboxy-terminal Diaphanous-autoregulatory domain (DAD), whose interaction stabilises an autoinhibited inactive conformation. Binding of active Rho releases DAD and activates the catalytic activity of mDia. Here, we report on the interaction of DAD with the regulatory N-terminus of mDia1 (mDia_N) and its release by RhoGTP. We have defined the elements required for tight binding and solved the three-dimensional structure of a complex between an mDia_N construct and DAD by X-ray crystallography. The core DAD region is an -helical peptide, which binds in the most highly conserved region of mDia_N using mainly hydrophobic interactions. The structure suggests a two-step mechanism for release of autoinhibition whereby RhoGTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that RhoGTP accelerates the dissociation of DAD from the mDia_NDAD complex.