CHARACTERIZATION OF THE ACTIVE-SITE OF P21 RAS BY ELECTRON-SPIN ECHO ENVELOPE MODULATION SPECTROSCOPY WITH SELECTIVE LABELING - COMPARISONSBETWEEN GDP AND GTP FORMS

Selectively labeled samples of human H- or N-ras p21 ligated to Mn(II) GDP or Mn(II)GMPPNP were studied by electron spin-echo envelope modula tion spectroscopy in order to define the protein environment around th e divalent metal. We incorporated [4-C-13]-labeled Asx into p21.Mn(II) GDP and found that the distance from the carboxyl C-13 of Asp57 to Mn( II) is approximately 4.1 angstrom. Our result is consistent with indir ect coordination of this residue to the metal. From a [2-H-2]Thr-label ed sample, we estimate that the distance from the Mn(II) ion to the H- 2 of Thr35 is at least 5.8 angstrom. Thus, the only protein or nucleot ide ligands to the metal appear to be Ser17 and the beta-phosphate of GDP, as previously reported [Larsen, R. G., Halkides, C. J., Redfield, A.G., & Singel, D.J. (1992b) J. Am. Chem. Soc. 114, 9608-9611]. In th e 5'-guanylylimido diphosphate (GMPPNP) form of p21, Thr35 has been re ported by X-ray crystallography to be a ligand of the metal via its hy droxyl group, and this residue appears to play a key role in the biolo gically important conformational change upon nucleotide substitution [ Pai, E. F., Krengel, U., Petsko, G., Goody, R. S., Kabsch, W., & Witti nghofer, A. (1990) EMBO J. 9, 2351-2359]. The ESEEM spectrum of p21.Mn (II)GMPPNP labeled with [2-H-2]Thr yields a Mn(II)-H-1 distance of 4.9 angstrom, a distance inconsistent with strong coordination. A sample of p21 in which the Thr residues were fully labeled with C-13 and N-15 yielded a value of 5.0 angstrom for the distance from Mn(II) to the a mide nitrogen of Thr35, while the C-13 signal is much smaller than exp ected if Thr35 were coordinated. A [N-15] serine/glycine-labeled sampl e gives a distance to the amide N-15 of Ser17 of 3.9 angstrom, consist ent with the X-ray structure; a [4-C-13]-labeled Asx sample of p21 giv es a distance of approximately 4 angstrom between Mn(II) and the label of Asp57, again implying indirect coordination. Both of these values are very similar to those found for the GDP form of the protein. The r esults for Thr35, however, reveal a structural difference between the GDP and GTP forms in the region of Thr35. In addition, the position of this residue is found to be different from the crystal structure and in a manner suggesting that the metal ligation of Thr35 does not drive the conformational change that accompanies nucleotide substitution.