Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily.
Although Ras residue phenylalanine-156 (F156) is strictly conserved in all members of the Ras superfamily of proteins, it is located outside of the consensus GDP/GTP-binding pocket. Its location within the hydrophobic core of Ras suggests that its strict conservation reflects a crucial role in structural stability. However, mutation of the equivalent residue (F157L) in the Drosophila Ras-related protein Rap results in a gain-of-function phenotype, suggesting an alternative role for this residue. Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Whereas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras. Further, Ras (156L) exhibited an extremely rapid off rate for bound GDP/GTP in vitro and showed increased levels of Ras.GTP in vivo. To determine the structural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused loss of contact with residues 6, 23, 55, and 79, resulting in disruption of secondary structure in alpha-helix 1 and in beta-sheets 1-5. These major structural changes contrast with the isolated alterations induced by oncogenic mutation (residues 12 or 61) that perturb GTPase activity, and instead, weaken Ras contacts with Mg2+ and its guanine nucleotide substrate and result in increased rates of GDP/GTP dissociation. Altogether, these observations demonstrate the essential role of this conserved residue in Ras structure and its function as a regulated GDP/GTP switch.
Quilliam
L A
LA
Department of Pharmacology, University of North Carolina, Chapel Hill 27599.
Zhong
S
S
Rabun
K M
KM
Carpenter
J W
JW
South
T L
TL
Der
C J
CJ
Campbell-Burk
S
S
D016475:3T3 Cells; D000818:Animals; D002471:Cell Transformation, Neoplastic; D019204:GTP-Binding Proteins / Q000378:metabolism* / Q000648:ultrastructure; D011905:Genes, ras; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism* / Q000648:ultrastructure; D013329:Structure-Activity Relationship
D006153:Guanosine Diphosphate; D010649:Phenylalanine; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D016283:Proto-Oncogene Proteins p21(ras)