High frequency (139.5 GHz) electron paramagnetic resonance characterization of Mn(II)-(H2O)-O-17 interactions in GDP and GTP forms of p21 ras

As a molecular switch, the ras protein p21 undergoes structural changes that couple recognition sites on the protein surface to the guanine nucleotide-divalent metal ion binding site, X-ray crystallographic studies of p21 suggest that coordination between threonine-35 and the divalent metal ion plays an important role in these conformational changes, Recent ESEEM studies of p21 in solution, however, place threonine-35 more distant ii om the metal and were interpreted as weak or indirect coordination of this residue, We report high frequency (139.5 GHz) EPR spectroscopy of p21 . Mn(II) complexes of two guanine nucleotides that probes the link between threonine-35 and the divalent metal ion, By analysis of high-frequency EPR spectra, we determine the number of water molecules in the first coordination sphere of the manganous ion to be four in p21 . Mn(II). GDP, consistent with prior low-frequency EPR and X-ray crystallographic studies, In the complex of p21 with a GTP analog, p21 . Mn(II). GMPPNP, we determine the hydration number to be 2, also consistent with crystal structures. This result rules out indirect coordination of threonine-35 in the solution structure of p21 . Mn(II). GMPPNP, and implicates direct, weak coordination of this residue as suggested by Halkides et al. [(1994) Biochemistry 33, 4019]. The O-17 hyperfine coupling constant of (H2O)-O-17 is determined as 0.25 mT in the GDP Corm and 0.28 mT in the GTP form, These values are similar to reported values for O-17-enriched aquo ligands and some phosphato ligands in Mn(II) complexes. The high magnetic field strength (4.9 T) employed in these 139.5 GHz EPR measurements leads to a narrowing of the Mn(II) EPR lines that facilitates the determination of O-17 hyperfine interactions.