Structural analysis of oncogenic H-Ras mutants G12A and G13A

grantor: University of TorontoAbout 30% of human cancers are due to point mutations in one of the H-, K-, and N-Ras genes. These small GTPases act as early switch molecules turning on and off cell proliferation processes in response to exocellular stimuli. Common mutations at Gly12, Gly13 and Gln61 prevent Ras from hydrolyzing GTP into GDP in the presence of a GAP (GTPase activating protein), prolonging the lifetime of the active state, therefore rendering the protein oncogenic. From previous mutational and crystallographic studies, we know that too big a side chain at position 12 would sterically hinder the correct positioning of the Gln61 side chain for catalysis. To answer the questions why G12A is oncogenic while G12P is not and why a small change from a hydrogen to a methyl group at Gly13 also makes the protein transforming, we studied crystal structures of G12A and G13A H-Ras. Free G12A/GDP was crystallized and subsequent modeling of its structure into the complex of H-Ras and p120GAPGRD showed that C ß of Ala12 occupied the same position as that of Pro12. The G13A/GDP structure revealed no sign of involvement of the 13th residue with any other part of the molecule or with GAP. Therefore, it appears that a full understanding of the influences of amino acid changes at these positions will require complex structures of H-Ras mutants and the catalytic domain of GAP.M.Sc