title: The frozen solution structure of p21 ras determined by ESEEM spectroscopy reveals weak coordination of Thr35 to the active site metal ion.
abstract: BACKGROUND
  The G protein p21 ras is a molecular switch in the signal transduction pathway for cellular growth and differentiation. Hydrolysis of tightly bound GTP alters the conformation of p21, terminating the signal. The coordination of the p21 residue Thr35 to Mg2+ in its active site, which has been observed in the crystal structure of p21 in complex with a GTP-analog GMPPNP but not with GDP, has been proposed to drive the conformational change accompanying nucleotide substitution and may have a role in the GTP hydrolysis reaction itself. However, previous electron spin-echo envelope modulation (ESEEM) studies of selectively 2H beta-threonine and 15N-threonine labeled p21.Mn2+ GMPPNP suggest that Thr35 only weakly coordinates the metal ion in the growth-active GTP-bound state of p21.
  
  
  RESULTS
  A 13C beta-Thr35 to Mn2+ distance of 4.3 +/- 0.2 A and a 15N epsilon-Lys16 to Mn2+ distance of 5.3 +/- 0.2 A were determined from ESEEM spectra of the selectively 13C beta-Thr and 15N epsilon-Lys labeled p21.Mn2+ GMPPNP frozen solution structure. The 13C beta-Thr35 to Mn2+ distance is greater than that (3.16 A) observed in the crystal structure. In contrast, the 15N epsilon-Lys16 to Mn2+ distance is in good agreement with the 5.1 A crystal structure distance.
  
  
  CONCLUSIONS
  The 13C beta of Thr35 is more distant from the active site Mn2+ in the frozen solution structure than in the crystal structure of p21.Mg2+ GMPPNP, indicating that Thr35 only weakly coordinates the metal ion in frozen solution. Thr35 coordination of the metal ion is therefore unlikely to drive the conformational change between GTP- and GDP-bound states of p21. Thr35 may be essential for GTPase-activating protein (GAP)-stimulated GTP hydrolysis and/or signal transduction for other reasons.
authors.affiliation: Department of Chemistry, Harvard University, Cambridge, MA 02138, USA.
authors.forename: C T
authors.initials: CT
authors.lastname: Farrar
authors.forename: C J
authors.initials: CJ
authors.lastname: Halkides
authors.forename: D J
authors.initials: DJ
authors.lastname: Singel
mesh_terms: D001665:Binding Sites; D002413:Cations, Divalent; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006165:Guanylyl Imidodiphosphate / Q000737:chemistry; D008345:Manganese / Q000737:chemistry*; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry*
chemical_list: D002413:Cations, Divalent; D013912:Threonine; D006165:Guanylyl Imidodiphosphate; D008345:Manganese; D016283:Proto-Oncogene Proteins p21(ras)