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Kinetic and Structural Analysis of the Mg2+ -binding Site of the Guanine Nucleotide-binding Protein p21 H-ras.

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  • Medizin
  • Ddc:610
  • Biology
  • Medicine


Kinetic and structural analysis of the Mg2+ -binding site of the guanine nucleotide-binding protein p21 H-ras Kinetic and Structural Analysis of the Mg2 + -binding Site of the Guanine Nucleotide-binding Protein p21 H-ras. (Received fot publkation, March 4, t992) Jacob Johnt:. Hans Rensland. Urne Schlichting, Ingrid Vetteri. Giao Domenico Borasio§, Roger S. Goody, and Alfred Wittinghofer From the Max-Planch-Institu//ilr Medi2in~'lChe For3chung, Abteilu1lll Biophysik, Jahnst~ 29, 6900 HeidelberB and the §Neurologi.'lche Klinik, UniversitiJt Munchen, Klinikum Großhadern, Marchionistra/k 15, 8(){)() Munclwn 70. Germany Tbe coordination and binding of tbe Mg'+ ion in the nucleotide~binding site of p21 have heen investigated using site-directed mutagenesis, kinetie methods. and phosphorous NMR. Mg'+ in the p21 *nucleotide'Mg2 + complex appears to be in rast equilibrium with the solvent. Tbe dissociation constant between Mg'+ aod tbe p21'GDP complex was determined to be 2.8 11M. It decreases 30- or 16-fold on substituting Ser-17 or Asp- 57 with alanine, respectively, whereas the T35A mu- tation has no effect. All three mutations influence the dissociation constants and the association and dissocia- tion rate constants of the interaction between guanine nucleotides and p21, but to a different degree, We conclude that Thr-35 is only complexed to Mgz+ in the GTP conforrnation and both Asp-57 and Ser-17 appear to be critical for both GDP and GTP binding. :n p NMR spectra of the GDP and Gpp(NH)p (guanosine-5'-(ß,"Y- imido)triphosphate) complexes of mutated p21 show a remarkable perturbation of the guanine nucleotide- binding site compared 10 wild-type protein, The mu- tant proteins show reduced GTPase rates, which are not stimulated by the GTPase-activating protein GAP, p21(S17A) has been reported to function just as p21(S17N) as a dominant negative inhibitor of normal p21. We find that it inhibits oncogenic p21-induced survival of prirnary neurons. Proteins invo

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