The frozen solution structure of p21 ras determined by ESEEM spectroscopy reveals weak coordination of Thr35 to the active site metal ion

Background: The G protein p21 res is a molecular switch in the signal transduction pathway for cellular growth and differentiation. Hydrolysis of tightly bound GTP alters the conformation of p21, terminating the signal. The coordination of the p21 residue Thr35 to Mg2+ in its active site, which has been observed in the crystal structure of p21 in complex with a GTP-analog GMPPNP but not with GDP, has been proposed to drive the conformational change accompanying nucleotide substitution and may have a role in the GTP hydrolysis reaction itself, However, previous electron spin-echo envelope modulation (ESEEM) studies of selectively H-2 beta-threonine and N-15-threonine labeled p21 . Mn(2+)GMPPNP suggest that Thr35 only weakly coordinates the metal ion in the growth-active GTP-bound state of p21. Results: A C-13 beta-Thr35 to Mn2+ distance of 4.3 +/- 0.2 Angstrom and a N-15 epsilon-Lys16 to Mn2+ distance of 5.3 +/- 0.2 Angstrom were determined from ESEEM spectra of the selectively C-13 beta-Thr and N-15 epsilon-Lys labeled p21 . Mn(2+)GMPPNP frozen solution structure. The C-13 beta-Thr35 to Mn2+ distance is greater than that (3.16 Angstrom) observed in the crystal structure. In contrast, the N-15 epsilon-Lys16 to Mn2+ distance is in good agreement with the 5.1 Angstrom crystal structure distance. Conclusions: The C-13 beta of Thr35 is more distant from the active site Mn2+ in the frozen solution structure than in the crystal structure of p21 . Mg(2+)GMPPNP, indicating that Thr35 only weakly coordinates the metal ion in frozen solution, Thr35 coordination of the metal ion is therefore unlikely to drive the conformational change between GTP-and GDP-bound states of p21. Thr35 may be essential for GTPase-activating protein (GAP)-stimulated GTP hydrolysis and/or signal transduction for other reasons.