Structural basis for CDC42 and RAC activation by the dual specificity GEF DOCK10

Dedicator of cytokinesis 10 (DOCK10) is a guanine nucleotide exchange factor (GEF) belonging to the DOCK family that activate RHO GTPases. DOCK10 controls amoeboid migration and IL-4 induced B cell activation. Recent structural studies of the catalytic DHR2 domains of DOCK-family GEFs DOCK2 and DOCK9 in the presence of nucleotide-free RAC and CDC42 respectively, have revealed a conserved mechanism of nucleotide exchange distinct from the large family of Dbl homology (DH) GEFs. Less is known from a biochemical and structural perspective, about DOCK10. We have determined the crystal structure of the apo-state of the DHR2 domain of DOCK10 (DOCK10DHR2), which provides the first structural model for the apo-state of a DHR2 domain for the entire DOCK family. It demonstrates the conformational changes within DOCK10DHR2 on engaging the GTPase, and associated induced conformational changes of the GTPase required to stimulate nucleotide release. We also report on the unexpected dual specificity of DOCK10DHR2 which directly interacts with RAC and CDC42 and induces the nucleotide exchange of both in vitro. This dual specificity is unique to DOCK10 within its evolutionarily related D subfamily. Structural studies of DOCK10DHR2 in complex with RAC revealed an intriguing 2:1 stoichiometry between DOCK10DHR2 and the GTPase, that differs from the canonical 1:1 stoichiometry for all other known DOCK-GTPase complexes. This was confirmed in solution using a variety of biophysical techniques. The identification of a novel mode of interaction between DOCK10DHR2 and the RAC GTPase provides new insights into how DOCK family GEFs discriminate between CDC42, RAC and RHOA.