Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras.

Details

Serval ID
serval:BIB_01C46CC85389
Type
Article: article from journal or magazin.
Collection
Publications
Title
Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras.
Journal
Journal of Biological Chemistry
Author(s)
John J., Rensland H., Schlichting I., Vetter I., Borasio G.D., Goody R.S., Wittinghofer A.
ISSN
0021-9258 (Print)
ISSN-L
0021-9258
Publication state
Published
Issued date
1993
Volume
268
Number
2
Pages
923-929
Language
english
Notes
Publication types: Journal ArticlePublication Status: ppublish
Abstract
The coordination and binding of the Mg2+ ion in the nucleotide-binding site of p21 have been investigated using site-directed mutagenesis, kinetic methods, and phosphorous NMR. Mg2+ in the p21.nucleotide.Mg2+ complex appears to be in fast equilibrium with the solvent. The dissociation constant between Mg2+ and the p21.GDP complex was determined to be 2.8 microM. It decreases 30- or 16-fold on substituting Ser-17 or Asp-57 with alanine, respectively, whereas the T35A mutation has no effect. All three mutations influence the dissociation constants and the association and dissociation rate constants of the interaction between guanine nucleotides and p21, but to a different degree. We conclude that Thr-35 is only complexed to Mg2+ in the GTP conformation and both Asp-57 and Ser-17 appear to be critical for both GDP and GTP binding. 31P NMR spectra of the GDP and Gpp(NH)p (guanosine-5'-(beta,gamma-imido)triphosphate) complexes of mutated p21 show a remarkable perturbation of the guanine nucleotide-binding site compared to wild-type protein. The mutant 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 primary neurons.
Keywords
Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Cells, Cultured, Chick Embryo, Cloning, Molecular, GTP Phosphohydrolases/chemistry, GTP Phosphohydrolases/genetics, Ganglia, Spinal/metabolism, Guanosine Diphosphate/metabolism, Guanylyl Imidodiphosphate/metabolism, Kinetics, Magnesium/metabolism, Magnetic Resonance Spectroscopy/methods, Molecular Sequence Data, Mutagenesis, Site-Directed, Neurons/metabolism, Oligodeoxyribonucleotides, PC12 Cells, Protein Conformation, Proto-Oncogene Proteins p21(ras)/chemistry, Proto-Oncogene Proteins p21(ras)/genetics, Recombinant Proteins/chemistry, Recombinant Proteins/isolation & purification, Transfection, X-Ray Diffraction
Pubmed
Web of science
Create date
13/01/2014 16:44
Last modification date
20/08/2019 12:24
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