Genetic and biochemical analysis of ras p21 structure.

We tested aspects of our model of the ras p21 structure using generic, biochemical, and immunologic approaches. First, we made a monoclonal antibody against a p21 region that is highly conserved and likely to be critical to p21 function. The antibody blocks p21 function in various cell systems. Its binding to p21 is completely blocked by guanine nucleotides, even though the region of p21 to which it binds does not seem to be part of the guanine nucleotide-binding site. We propose that the conformation of this critical region is modulated by nucleotide binding. Another interesting region of p21 includes amino acids 116 and 119, which seem to confer, in part, the specificity of p21 for guanine nucleotides. We made a series of mutants in this region and tested their ability to bind GTP, and such related purine nucleotides as XTP and diaminopurine nucleoside triphosphate. We were able to refine our model for guanine nucleotide interaction with p21 and to create mutant proteins with altered specificity for purine nucleotides. Finally, we tested rates of autophosphorylation of six position 12 mutants and conclude that amino acid 12 affects the positioning of bound nucleotides relative to sequences around amino acid 59.