Molecular mechanism of self-activation induced by the intrinsically disordered region in Rac1b: Structural and...

Rac1b is a splicing variant of the RAC1 gene, characterized by a 19-amino-acid (19AA) insertion between residues 75 and 76. Overexpression of Rac1b has been observed in various cancers, establishing it as a potential target for anticancer therapies. Similar to Rac1, Rac1b functions as a GTPase, cycling between an active GTP-bound state and an inactive GDP-bound state. However, due to the presence of the 19AA insertion, Rac1b undergoes GTP/GDP exchange independently of guanine nucleotide exchange factors (GEFs). Using combined molecular dynamics simulations and experimental approaches, we demonstrate that this insertion enhances the flexibility of the critical Switch I and Switch II regions, weakens Rac1b's interaction with Mg²⁺, and reduces its GDP-binding affinity. Mechanistically, the 19AA insertion disrupts Switch I-II interactions, inducing a destabilizing "see-saw" dynamic in Switch I that facilitates rapid GDP dissociation. This mechanism resembles GEF-mediated Rac1 activation, suggesting that the 19AA insertion functionally mimics GEF activity. Furthermore, five distinct conformational sub-states were identified during Rac1b inactivation, revealing cryptic small-molecule binding pockets. These findings provide deeper insights into the role of intrinsically disordered regions in protein function and offer a structural foundation for the rational design of Rac1b-targeted inhibitors.