Assessing the Mechanism of Rac1b: An All-Atom Simulation Study of the Alternative Spliced Variant of Rac1 Small Rho GTPase

The Rho GTPase family plays a key role in cell migration, cytoskeletal dynamics, and intracellular signaling. Rac1 and its splice variant Rac1b, characterized by the insertion of an Extraloop, are frequently associated with cancer. These small GTPases switch between an active GTP-bound state and an inactive GDP-bound state, a process that is regulated by specific protein modulators. Among them, the Guanine nucleotide exchange factor (GEF) protein DOCK5 specifically targets Rho GTPases, promoting their activation by facilitating the exchange of GDP for GTP. In this study, we performed cumulative 10-μs-long all-atom molecular dynamics simulations of Rac1 and Rac1b, in isolation and in complex with DOCK5 and ELMO1, to investigate the impact of the Rac1b Extraloop. Our findings reveal that this Extraloop decreases the GDP residence time as compared to Rac1, mimicking the effect of accelerated GDP/GTP exchange induced by DOCK5. Furthermore, both Rac1b Extraloop and the ELMO1 protein stabilize the GTPase/DOCK5 complex, contributing to facilitate GDP dissociation. This shifts the balance between the GPT- and GDP-bound state of Rac1b toward the active GTP-bound state, sending a prooncogenic signal. Besides broadening our understanding of the biological functions of small Rho GTPases, this study provides key information to exploit a previously unexplored therapeutic niche to counter Rac1b-associated cancer.