Structural and biophysical insights into the role of the insert region in Rac1 function

A 13 amino acid insertion that forms a short 3₁₀ helix between β-strand 5 and α-helix 4 is a distinguishing feature among most members of the Rho family of GTPases, yet the precise role of this region in signal transduction is poorly understood. Previous in vivo functional studies have implicated the insert region of RhoA, Rac1, and Cdc42 to be important for cell transformation, regulation of the actin cytoskeleton, controlling DNA synthesis, and in the activation of downstream targets. In Rac1, our recent biological studies have suggested that the insert is important for SRF activation and the formation of lamellipodia but is dispensable for all other cellular functions of this protein. In the studies reported herein, we have characterized the effect of the insert deletion on Rac1 structure, thermodynamic stability, and the kinetics of nucleotide association. These in vitro studies help clarify biological data and provide further insights as to the role of this 13 amino acid region in modulating Rac1 function. The studies reveal that deletion of the insert has no effect on Rac1 structure and causes only a marginal (∼0.8 kcal/mol) decrease in the ΔG^fold of Rac1·GDP·Mg²⁺. The intrinsic rate of nucleotide dissociation of Rac1·Δ^ins is decreased by about 1.5-fold compared to that of wild type, and a 3-fold increase in the GEF (Vav2)-mediated exchange rate is observed. In addition, deletion of the insert does not change the K_D for the interaction of Rac1 with GDI, and similar to that previously observed for Cdc42, no inhibition of GDP dissociation is observed for the deletion mutant relative to that for the native protein. Taken together, the structural and biochemical studies reported here are consistent with our biological data reported previously and suggest that the most likely role of the insert region must be to serve as a binding interface for downstream effectors, particularly those important for actin regulation.