CHARACTERIZATION OF THE ACTIVE-SITE OF P21 RAS BY ELECTRON-SPIN ECHO ENVELOPE MODULATION SPECTROSCOPY WITH SELECTIVE LABELING - COMPARISONSBETWEEN GDP AND GTP FORMS
Cj. Halkides et al., CHARACTERIZATION OF THE ACTIVE-SITE OF P21 RAS BY ELECTRON-SPIN ECHO ENVELOPE MODULATION SPECTROSCOPY WITH SELECTIVE LABELING - COMPARISONSBETWEEN GDP AND GTP FORMS, Biochemistry, 33(13), 1994, pp. 4019-4035
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.