The three dimensional structure of the Harvey -Ras G12V V45E mutant complexed with guanosine diphosphate and the nonhydrolyzable analog of guanosine triphosphate, guanylylimido diphosphat

Among the most common genetic lesions contributing to the formation of human malignancies are activating mutations in the ras oncogenes. The Ras proteins, H, N and K, encode homologous 21-kDa proteins that exist in two conformations, a GDP-bound inactive state and a GTP-bound active state. Activated Ras influences cellular behavior by interacting with, and controlling, target enzymes, known as effectors. Among the most common Ras mutations that produce a transformed phenotype is Ras G12V.;Rap1a is a 21-kDa guanine-nucleotide-binding protein originally identified as a suppresser of K-Ras mediated transformation in NIH 3T3 cells. It has been hypothesized that these suppression effects occur via competition with Ras for protein effectors. Consistent with this hypothesis, Ras and Rap1a exhibit a high degree of sequence homology. Variants incorporating nonconserved positions of Rap1a into positions in the transforming H-Ras G12V gene, were examined in a focus-formation assay to determine their biological potency. The mutant H-Ras protein G12V, V45E, was found to be one thousand-fold less efficient than H-Ras G12V in focus formation.;X-Ray crystallographic studies were initiated to determine if the G12V and V45E mutations in the H-Ras protein caused a structural change that rendered the protein incapable of interacting with its effectors. No gross structural changes were observed in either the GDP-bound or GMPPNP-bound H-Ras G12V V45E protein. Therefore, it is believed that the biological activity exhibited by the H-Ras G12V V45E mutant is due to the change in nature of the side chain that accompanies the V45E mutation.