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. Hydrolys is of tightly bound GTP alters the conformation of p21, terminating th e signal. The coordination of the p21 residue Thr35 to Mg2+ in its act ive site, which has been observed in the crystal structure of p21 in c omplex 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, pr evious electron spin-echo envelope modulation (ESEEM) studies of selec tively H-2 beta-threonine and N-15-threonine labeled p21 . Mn(2+)GMPPN P suggest that Thr35 only weakly coordinates the metal ion in the grow th-active GTP-bound state of p21. Results: A C-13 beta-Thr35 to Mn2+ d istance of 4.3 +/- 0.2 Angstrom and a N-15 epsilon-Lys16 to Mn2+ dista nce 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+)GMP PNP 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 agreem ent 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 f rozen 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 unl ikely to drive the conformational change between GTP-and GDP-bound sta tes of p21. Thr35 may be essential for GTPase-activating protein (GAP) -stimulated GTP hydrolysis and/or signal transduction for other reason s.