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

The\nRho GTPase family plays a key role in cell migration, cytoskeletal\ndynamics, and intracellular signaling. Rac1 and its splice variant\nRac1b, characterized by the insertion of an Extraloop, are frequently\nassociated with cancer. These small GTPases switch between an active\nGTP-bound state and an inactive GDP-bound state, a process that is\nregulated by specific protein modulators. Among them, the Guanine\nnucleotide exchange factor (GEF) protein DOCK5 specifically targets\nRho GTPases, promoting their activation by facilitating the exchange\nof GDP for GTP. In this study, we performed cumulative 10-μs-long\nall-atom molecular dynamics simulations of Rac1 and Rac1b, in isolation\nand in complex with DOCK5 and ELMO1, to investigate the impact of\nthe Rac1b Extraloop. Our findings reveal that this Extraloop decreases\nthe GDP residence time as compared to Rac1, mimicking the effect of\naccelerated GDP/GTP exchange induced by DOCK5. Furthermore, both Rac1b\nExtraloop and the ELMO1 protein stabilize the GTPase/DOCK5 complex,\ncontributing to facilitate GDP dissociation. This shifts the balance\nbetween the GPT- and GDP-bound state of Rac1b toward the active GTP-bound\nstate, sending a prooncogenic signal. Besides broadening our understanding\nof the biological functions of small Rho GTPases, this study provides\nkey information to exploit a previously unexplored therapeutic niche\nto counter Rac1b-associated cancer.