Abstract

Selectively labeled samples of human H- or N-ras p21 ligated to MnIIGDP or MnIIGMPPNP were studied by electron spin-echo envelope modulation spectroscopy in order to define the protein environment around the divalent metal. We incorporated [4-13C]-labeled Asx into p21.MnIIGDP and found that the distance from the carboxyl 13C of Asp57 to MnII is approximately 4.1 A. Our result is consistent with indirect coordination of this residue to the metal. From a [2-2H]Thr-labeled sample, we estimate that the distance from the MnII ion to the 2H of Thr35 is at least 5.8 A. Thus, the only protein or nucleotide 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 the 5'-guanylylimido diphosphate (GMPPNP) form of p21, Thr35 has been reported by X-ray crystallography to be a ligand of the metal via its hydroxyl group, and this residue appears to play a key role in the biologically important conformational change upon nucleotide substitution [Pai, E. F., Krengel, U., Petsko, G., Goody, R. S., Kabsch, W., & Wittinghofer, A. (1990) EMBO J. 9, 2351-2359]. The ESEEM spectrum of p21.MnIIGMPPNP labeled with [2-2H]Thr yields a MnII-2H distance of 4.9 A, a distance inconsistent with strong coordination. A sample of p21 in which the Thr residues were fully labeled with 13C and 15N yielded a value of 5.0 A for the distance from MnII to the amide nitrogen of Thr35, while the 13C signal is much smaller than expected if Thr35 were coordinated. A [15N]serine/glycine-labeled sample gives a distance to the amide 15N of Ser17 of 3.9 A, consistent with the X-ray structure; a [4-13C]-labeled Asx sample of p21 gives a distance of approximately 4 A between MnII 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 results 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.