Mutations of Ha-ras p21 that define important regions for the molecular mechanism of the SDC25 C-domain, a guanine nucleotide dissociation stimulator.
The SDC25 C-domain is a very active guanine nucleotide dissociation stimulator (GDS) isolated from Saccharomyces cerevisiae which acts equally well on Ha-ras p21 and yeast RAS2. These properties make the SDC25 C-domain a suitable tool to study the basic mechanism of a GDS. The action of the SDC25 C-domain was analysed by mutation of structurally important regions of p21. Substitutions that influence the coordination of Mg2+.GDP or the interaction of the guanine ring were found to stimulate the intrinsic dissociation of GDP and suppress the action of the SDC25 C-domain. No relevant effects were observed with mutations in the phosphate binding loop L1 or by deleting the last 23 C-terminal residues of p21. Substitutions in the switch region 1 (loop L2) and 2 (loop L4) of p21 strongly impaired the action of this GDS; however, we show that this effect is not related to a decreased affinity of the SDC25 C-domain for the mutated p21. No functional competition could be found between this GDS and the catalytic domain of the human GTPase activating protein (GAP). This indicates that GDS and GAP bind to different sites of the p21.nucleotide complex, even though the same mutations in loops L2 and L4 regions affect the activity of both effectors. Since these two regions appear not to be involved directly in the interaction with GDS, we conclude that the negative effect induced by their mutation is related to their function as switches of selective conformations during the GDP to GTP exchange reaction catalysed by GDS.
Mistou
M Y
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S.D.I. no. 61840 du C.N.R.S., Laboratoire de Biochimie, Ecole Polytechnique, Palaiseau, France.
Jacquet
E
E
Poullet
P
P
Rensland
H
H
Gideon
P
P
Schlichting
I
I
Wittinghofer
A
A
Parmeggiani
A
A
D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D005798:Genes, Bacterial; D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008968:Molecular Conformation; D009154:Mutation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D020668:rap GTP-Binding Proteins
D005656:Fungal Proteins; D006150:Guanine Nucleotides; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras); D020668:rap GTP-Binding Proteins