Teeter-Totter-Like Redistribution of Switch I: Integrating Simulation and Experiment to Uncover the Molecular Basis...

Rac1 is a small GTPase of the Rho family. It is a central regulator of cytoskeletal remodeling, cell adhesion, migration, and secretion. Dysregulation of Rac1 activity drives tumorigenesis, with the cancer-associated P29S mutation ranking as the third most frequently mutated proto-oncogene in sun-exposed melanoma. P29S is a prototypical gain-of-function, fast-cycling variant that destabilizes GDP binding and accelerates nucleotide exchange. Another cancer-associated mutant, F28L, produces cellular phenotypes strikingly similar to those of P29S yet has been proposed to act through a distinct local structural perturbation, raising the question of whether they share a common activation mechanism. Defining this shared mechanism is critical for understanding Rac1-driven oncogenesis and for guiding the rational design of inhibitors that effectively target these activating mutations. Here, integrating molecular dynamics simulations, potential of mean force calculations, and biochemical assays, we show that P29S, F28L, and the structurally distinct L160C mutant all weaken GDP binding while enhancing GTP affinity─revealing an oncogenic mechanism beyond fast cycling. Structural dynamic analyses uncover a shared "teeter-totter-like" redistribution of the Switch I region, in which destabilization of its N-terminal hydrophobic core is coupled to reinforcement of its C-terminal contact network. This dynamic shift strengthens effector binding, as validated by GTPase pull-down assays, and is also reproduced by other cancer-associated mutants in Rac1 (I21S, P34S, D57E). Our findings identify the β6-α5 loop as an allosteric hub linking nucleotide binding to effector engagement and suggest that rebalancing the Switch I region may provide a therapeutic strategy to counteract Rac1-driven oncogenic signaling.