Three-dimensional structures and properties of a transforming and a nontransforming glycine-12 mutant of p21H-ras.
The three-dimensional structures and biochemical properties of two mutants of the G-domain (residues 1-166) of p21H-ras, p21 (G12D) and p21 (G12P), have been determined in the triphosphate-bound form using guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp). They correspond to the most frequent oncogenic and the only nononcogenic mutation of Gly-12, respectively. The G12D mutation is the only mutant analyzed so far that crystallizes in a space group different from wild type, and the atomic model of the protein shows the most drastic changes of structure around the active site as compared to wild-type p21. This is due to the interactions of the aspartic acid side chain with Tyr-32, Gln-61, and the gamma-phosphate, which result in reduced mobility of these structural elements. The interaction between the carboxylate group of Asp-12 and the gamma-phosphate is mediated by a shared proton, which we show by 31P NMR measurements to exist in solution as well. The structure of p21 (G12P) is remarkably similar to that of wild-type p21 in the active site, including the position of the nucleophilic water. The pyrrolidine ring of Pro-12 points outward and seems to be responsible for the weaker affinity toward GAP (GTPase-activating protein) and the failure of GAP to stimulate GTP hydrolysis.
Franken
S M
SM
Abteilung Biophysik, Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Scheidig
A J
AJ
Krengel
U
U
Rensland
H
H
Lautwein
A
A
Geyer
M
M
Scheffzek
K
K
Goody
R S
RS
Kalbitzer
H R
HR
Pai
E F
EF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D017124:Conserved Sequence; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D011905:Genes, ras; D005998:Glycine; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008322:Mammals; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras); D008274:Magnesium; D005998:Glycine