High-Frequency 94 GHz ENDOR Characterization of the Metal Binding Site in Wild-Type Ras·GDP and Its Oncogenic Mutant G12V in Frozen Solution

Abstract

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.

W-band31P-ENDOR on the high-affinity Mn2+ binding site in the minimal and tertiary stabilized hammerhead ribozymes

“…Such a large splitting can be exptained by a large isotropic 3lp hyperfine coupling of about 8.4 MHz but not by a large hyperfine anisotropy. Even in an inner-sphere bound fashion, the manganese-phosphorus distance cannot be much closer than 0.34 nm, which would correspond to a [T~[ of only 0.8 MHz as also found for the PI3 of the Mn2+-ADP [22] and the human p21ras-Mn 2+-GDP [25] complexes. On the other hand, assuming such an inner-sphere coordination results in a dipolar tensor width of I T• 3/2 = 1.2 MHz, which lies within the observed line width of about 2.2 MHz and agrees with the [Aiso(31p)[ of about 8.4 MHz.…”

Quantitative characterization of the Mn2+ complexes of ADP and ATPγS by W-band ENDOR

“…In some tases 14N couplings were detected as well, revealing coordination to the protein [5,13]. Recently, also high-field ~70 ENDOR was used to identify water ligands [7]. Table 1 shows that the coordination of two phosphates is revealed when two different 3lp hyperfine couplings are observed, like in H+ATPase-Mn 2+-AMPPNP [14].…”

“…A family of related enzymes are those involving the nucleotides GTP (guanosine triphosphate) and GDP (guanosine diphosphate). Examples are the protein Ras, which plays a central role asa molecular switch in cellular signal transduction [7], and the…”

“…A family of related enzymes are those involving the nucleotides GTP (guanosine triphosphate) and GDP (guanosine diphosphate). Examples are the protein Ras, which plays a central role asa molecular switch in cellular signal transduction [7], and the + Mn2~ GDP-mannose mannosyl hydrolase (GDPMH), which catalyzes the hydrolysis of GDP-cc-D-sugars to yield the 13-D-sugar and GDP [8]. Divalent metal binding to nucleotides is also highly relevant in RNA catalysis, where again Mn 2+ has been used to illuminate the role of the metal ions [9,10].…”

Spin-correlated radical pairs: the differential effect in high-field ENDOR spectra

“…These approaches yield unique information on dynamics, structure and electron transfer pathways of the photosynthetic proteins [1][2][3]. Concomitantly, the associated HF method of electron-nuclear double resonance (ENDOR), which measures electron-nuclear hyperfine interactions (HFI), has the potential to directly probe the structure of cofactors and their local protein environments [1,2,[11][12][13].…”