Biological and structural characterization of a Ras transforming mutation at the phenylalanine-156 residue, which is conserved in all members of the Ras superfamily

Lawrence A. Quilliam, Sheng Zhong, Kelly M. Rabun, John W. Carpenter, Terri L. South, Channing J. Der, Sharon Campbell-Burk

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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 α-helix 1 and in β-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.

Original languageEnglish (US)
Pages (from-to)1272-1276
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number5
StatePublished - Feb 28 1995



  • NMR
  • guanine nucleotide binding

ASJC Scopus subject areas

  • General

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