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 hydr ophobic core of Ras suggests that its strict conservation reflects a c rucial role in structural stability, However, mutation of the equivale nt residue (F157L) in the Drosophila Ras-related protein Rap results i n a gain-of-function phenotype, suggesting an alternative role for thi s residue, Therefore, we have introduced an F156L mutation into Ras to evaluate the role of this residue in Ras structure and function. Wher eas introduction of this mutation activated the transforming potential of wild-type Ras, it did not impair that of oncogenic Ras, Further, R as(156L) exhibited an extremely rapid off rate for bound GDP/GTP in vi tro and showed increased levels of Ras GTP in vivo. To determine the s tructural basis for these altered properties, we used high-resolution nuclear magnetic resonance spectroscopy. The F156L mutation caused los s 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 induce d by oncogenic mutation (residues 12 or 61) that perturb GTPase activi ty, and instead, weaken Ras contacts with Mg2+ and its guanine nucleot ide substrate and result in increased rates of GDP/GTP dissociation. A ltogether, these observations demonstrate the essential role of this c onserved residue in Ras structure and its function as a regulated GDP/ GTP switch.