Rac small GTPases and their GEFs of the DOCK family are pivotal checkpoints in development, autoimmunity and bone homeostasis, and their abnormal regulation is associated to diverse pathologies. Small molecules that inhibit their activities are therefore needed to investigate their functions. Here, we characterized the mechanism of inhibition of human DOCK5 by C21, a small molecule that inhibits mouse Dock5 in cells and blocks bone degradation in mice models of osteoporosis. We showed that the catalytic DHR2 domain of DOCK5 has a high basal GEF activity in the absence of membranes which is not regulated by a simple feedback loop. C21 blocks this activity in a non-competitive manner and is specific for DOCK5. In contrast, another Dock inhibitor, CPYPP, inhibits both DOCK5 and an unrelated GEF, Trio. To gain insight into structural features of the inhibitory mechanism of C21, we used SAXS analysis of DOCK5DHR2 and crystallographic analysis of unbound Rac1-GDP. Together, these data suggest that C21 takes advantage of intramolecular dynamics of DOCK5 and Rac1 to remodel the complex into an unproductive conformation. Based on this allosteric mechanism, we propose that diversion of intramolecular dynamics is a potent mechanism for the inhibition of multidomain regulators of small GTPases.

Bacterial kinesin light chain is a TPR domain-containing protein encoded by the bklc gene, which co-localizes with the bacterial tubulin (btub) genes in a conserved operon in Prosthecobacter. Btub heterodimers show high structural homology with eukaryotic tubulin and assemble into head-to-tail protofilaments. Intriguingly, Bklc is homologous to the light chain of the microtubule motor kinesin and could thus represent an additional eukaryotic-like cytoskeletal element in bacteria. Using biochemical characterization as well as cryo-electron tomography we show here that Bklc interacts specifically with Btub protofilaments, as well as lipid vesicles and could thus play a role in anchoring the Btub filaments to the membrane protrusions in Prosthecobacter where they specifically localize in vivo. This work sheds new light into possible ways in which the microtubule cytoskeleton may have evolved linking precursors of microtubules to the membrane via the kinesin moiety that in today’s eukaryotic cytoskeleton links vesicle-packaged cargo to microtubules.