Allosteric inhibition of the guanine nucleotide exchange factor DOCK5 by a small molecule

Yann Ferrandez,Wenhua Zhang,François Peurois,Anne Blangy,Jacqueline Cherfils,Lurlène Akendengué,Mahel Zeghouf

doi:10.1038/s41598-017-13619-2

Abstract

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.

Highlights

DOCK proteins activate Rho family GTPases to regulate development, autoimmunity and bone homeostasis and they are involved in cancer and severe developmental and immune-related diseases
We found that unbound DOCK5DHR2 resembles the dimeric structure of Dock2DHR2 and that it displays conformational dynamics that diminishes upon binding of Rac[1]
The kinetics of nucleotide exchange was monitored in solution in the presence of catalytic amounts of DOCK5DHR2, by following the decay in fluorescence associated to the replacement of Mant-GDP by GTP

Summary

Introduction

DOCK (dedicator of cytokinesis) proteins activate Rho family GTPases to regulate development, autoimmunity and bone homeostasis and they are involved in cancer and severe developmental and immune-related diseases (reviewed in refs[1,2,3]). Crystal structures of DHR2/GTPase complexes have been solved for Dock9/Cdc[424] and Dock2/Rac[15,6] and for a truncated DHR2 domain from Dock[8] in complex with Cdc[427] These structures identified key features of DHR2 domains and provided a description of the steps involved in GDP dissociation and GTP reloading. Our data suggest a model in which C21 takes advantage of the intramolecular dynamics of DOCK5 and Rac1-GDP to remodel the complex in a conformation that cannot proceed to GDP dissociation Based on this mechanism, we propose that diversion of intramolecular dynamics can be exploited by chemical compounds to remodel protein complexes into unproductive conformations, and has the potential for a variety of applications to the inhibition of GEFs and multidomain proteins in diseases

Methods

Results

Conclusion