Dynamic properties of the Ras switch I region and its importance for binding to effectors

We have investigated the dynamic properties of the switch I region of the G TP-binding protein Ras by using mutants of Thr-35, an invariant residue nec essary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a re duced affinity to Ras effector proteins without Thr-35 being involved in an y interaction. The structure of Ras(T35S)-GppNHp was determined by x-ray cr ystallography. Whereas the overall structure is very similar to wildtype, r esidues from switch I are completely invisible, indicating that the effecto r loop region is highly mobile. P-31-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) c orresponds to the structure found in the complex with the effecters. P-31-N MR spectra of pas mutants (T35S) and (T35A) in the GppNHp form show that th e equilibrium is shifted such that they occur predominantly in the nonbindi ng conformation (state 1). On addition of pas effecters, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-ste p binding reaction of wildtype and (T35S)Ras with effecters requires the ex istence of a rate-limiting isomerization step, which is not observed with T 35A, The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effecters. The dynamics of the swit ch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.