High-frequency 94 GHz ENDOR characterization of the metal binding site in wild-type Ras x GDP and its oncogenic mutant G12V in frozen solution.
The guanine nucleotide binding protein Ras plays a central role as molecular switch in cellular signal transduction. Ras cycles between a GDP-bound "off" state and a GTP-bound "on" state. Specific oncogenic mutations in the Ras protein are found in up to 30% of all human tumors. Previous 31P NMR studies had demonstrated that in liquid solution different conformational states in the GDP-bound as well as in the GTP-bound form coexist. High-field EPR spectroscopy of the GDP complexes in solution displayed differences in the ligand sphere of the wild-type complex as compared to its oncogenic mutant Ras(G12V). Only three water ligands were found in the former with respect to four in the G12V mutant [Rohrer, M. et al. (2001) Biochemistry 40, 1884-1889]. These differences were not detected in previous X-ray structures in the crystalline state. In this paper, we employ high-frequency electron nuclear double resonance (ENDOR) spectroscopy to probe the ligand sphere of the metal ion in the GDP-bound state. This technique in combination with selective isotope labeling has enabled us to detect the resonances of nuclei in the first ligand sphere of the ion with high spectral resolution. We have observed the 17O ENDOR spectra of the water ligands, and we have accurately determined the 17O hyperfine coupling with a(iso) = -0.276 mT, supporting the results of previous line shape analysis in solution. Further, the distinct resonances of the alpha-, beta-, and gamma-phosphorus of the bound nucleotides are illustrated in the 31P ENDOR spectra, and their hyperfine tensors lead to distances in agreement with the X-ray structures. Finally, 13C ENDOR spectra of uniformly 13C-labeled Ras(wt) x GDP and Ras(G12V) x GDP complexes as well as of the Ras(wt) x GppNHp and the selectively 1,4-13C-Asp labeled Ras(wt) x GDP complexes have revealed that in frozen solution only one amino acid is ligated to the ion in the GDP state, whereas two are bound in the GppNHp complex. Our results suggest that a second conformational state of the protein, if correlated with a different ligand sphere of the Mn2+ ion, is not populated in the GDP form of Ras at low temperatures in frozen solution.
Bennati
M
M
Institute of Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance, J. W. Goethe University of Frankfurt, D-60439 Frankfurt, Germany. bennati@chemie.uni-frankfurt.de
Hertel
M M
MM
Fritscher
J
J
Prisner
T F
TF
Weiden
N
N
Hofweber
R
R
Spörner
M
M
Horn
G
G
Kalbitzer
H R
HR
D001665:Binding Sites; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005615:Freezing; D005998:Glycine / Q000235:genetics; D006153:Guanosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D009857:Oncogenes; D010758:Phosphorus / Q000737:chemistry / Q000378:metabolism; D012996:Solutions / Q000737:chemistry; D014633:Valine / Q000235:genetics; D014867:Water / Q000737:chemistry; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D008670:Metals; D009711:Nucleotides; D012996:Solutions; D014867:Water; D006153:Guanosine Diphosphate; D010758:Phosphorus; D006165:Guanylyl Imidodiphosphate; D018631:ras Proteins; D014633:Valine; D005998:Glycine