The regulation of mDia1 by autoinhibition and its release by Rho•GTP

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 carboxyterminal 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 Rho center dot GTP. We have defined the elements required for tight binding and solved the three-dimensional structure of a complex between an mDiaN construct and DAD by X-ray crystallography. The core DAD region is an a-helical peptide, which binds in the most highly conserved region of mDiaN using mainly hydrophobic interactions. The structure suggests a two-step mechanism for release of autoinhibition whereby Rho center dot GTP, although having a partially nonoverlapping binding site, displaces DAD by ionic repulsion and steric clashes. We show that Rho center dot GTP accelerates the dissociation of DAD from the mDia(N)center dot DAD complex.