Switch II region

Fig. 15. Conformational differences between Ras GTP Mg2+ and Ras GDP Mg2+ (PDB ID 4Q21) complexes. The a-helices are denoted A1-A5, the p-sheets are denoted B1-B6, and the loops are denoted G1-G5. The Ras GTP complex is approximated by the cocrystallization of a nonhydrolyzable GTP analog containing a methylene linkage between the p- and y-phosphates (PDB ID 5P21). The GTPase activity of Ras prevents crystallization of the complex with GTP. Note the significant conformational differences within the Switch I and Switch II regions between the two structures. The transition between these two states occurs upon GTP hydrolysis.

The switch II region (residues 59-67) includes a conserved DXXG motif. Gly60 of this motif forms an H-bridge to the y-phosphate. Switch II also contains the cataly-tically essential Gln61 residue and is involved in the interaction with GTPase-activat-ing proteins. 

The structural difference between the active GTP-state and the inactive GDP-state of the Ras protein is primarily confined to the switch I and switch II regions. The conformational change can be described best by a loaded spring mechanism (see Fig. 5.22), where the two switch regions are fixed by the y-phosphate of GTP in a tense state. Upon GTP-hydrolysis, the two switch regions are allowed to relax into the GDP-spe-cific position. Thereby, the coordination of switch I to the y-phosphate and to Mg2 is lost as well as the interaction of the conserved Gly60 in switch II with the y-phosphate. 

The effect of oncogenic mutations at position 61 of the switch II region can also be explained using the Ras-GAP complex. Gln61 has a central function in GTP hydrolysis in that it contacts and coordinates the hydrolytic water molecule and the O-atom of y-phosphate of GTP and thus stabilizes the transition state. Amino acids with other side chains apparently cannot fulfill this function, as shown by the oncogenic effect of Gln61 mutants in which Gln61 is replaced by other amino acids (other than Glu). 

The production of preparative amounts of homogeneously adenylylated Rabl allows detailed binding studies of the modified protein with regulatory factors (that are GEF, GAP, GDI) and downstream effectors. As the site of adenylylation is situated in the highly important switch II region, this modification would hypothetically interfere with the binding of most proteins. The facile production of the modified protein ensures that even interactions of moderate-to-weak affinity can be quantified. 

Small G-proteins contain two regions named switch I and switch II, which undergo a binary conformational change upon nucleotide exchange and GTP hydrolysis (Vetter and Wittinghofer, 2001). The switch II region of Arf and Sar contains a conserved tryptophan residue, which acts as an intrinsic fluorescent probe of the protein conformation. The intrinsic fluorescence of Arfl and Sari increases (by +100% and +200%, respectively) when GDP is replaced by GTP. Tryptophan fluorescence is thus a convenient way to follow the activation-inactivation cycle of these small G-proteins in real-time (Antonny et al, 1997,2001 Bigay et al., 2003 Futai et al, 2004). 

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