MUTAGENESIS OF THE H-RAS P21 AT GLYCINE-60 RESIDUE DISRUPTS GTP-INDUCED CONFORMATIONAL CHANGE

The function of Gly-60, the conserved glycine in the DXXG domain of v- H-ras, was examined by site-directed mutagenesis. It was found that wh ile the G60A (Gly-60 to Ala substitution) mutation has little effect o n the interaction of H-ras with guanine nucleotides, it completely abo lishes the biological activity of v-H-ras. The G60A mutation also exer ts little effect on the interaction of H-ras with SDC25C (a guanine nu cleotide exchange factor) and GAP. However, the G60A mutation does low er the ability of H-ras to bind Raf. GTP induces an enhancement of flu orescence emission in complexes consisting of H-ras and the fluorescen t dye 8-anilino-1-naphthalenesulfonic acid. This enhancement is blocke d by the G60A mutation. On the basis of these observations, we propose that the GTP-induced conformational change of H-ras, a process requir ed for H-ras activities, is impaired by the G60A mutation.