| Abstract
| - A 13 amino acid insertion that forms a short 310 helix between β-strand 5 and α-helix 4 is adistinguishing feature among most members of the Rho family of GTPases, yet the precise role of thisregion in signal transduction is poorly understood. Previous in vivo functional studies have implicatedthe insert region of RhoA, Rac1, and Cdc42 to be important for cell transformation, regulation of theactin cytoskeleton, controlling DNA synthesis, and in the activation of downstream targets. In Rac1, ourrecent biological studies have suggested that the insert is important for SRF activation and the formationof lamellipodia but is dispensable for all other cellular functions of this protein. In the studies reportedherein, 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 providefurther insights as to the role of this 13 amino acid region in modulating Rac1 function. The studiesreveal that deletion of the insert has no effect on Rac1 structure and causes only a marginal (∼0.8 kcal/mol) decrease in the ΔGfold of Rac1·GDP·Mg2+. The intrinsic rate of nucleotide dissociation of Rac1·Δinsis 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 KD for theinteraction of Rac1 with GDI, and similar to that previously observed for Cdc42, no inhibition of GDPdissociation is observed for the deletion mutant relative to that for the native protein. Taken together, thestructural and biochemical studies reported here are consistent with our biological data reported previouslyand suggest that the most likely role of the insert region must be to serve as a binding interface fordownstream effectors, particularly those important for actin regulation.
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