ONCOGENIC AMINO-ACID SUBSTITUTIONS IN THE INHIBITORY RAP-1A PROTEIN CAUSE IT TO ADOPT A RAS-P21-LIKE CONFORMATION AS COMPUTED USING MOLECULAR-DYNAMICS

rap-1A is a membrane-bound G-protein in the ras superfamily that, like the ras-p21 protein, is activated by binding GTP in place of GDP When activated, however, this protein inhibits the action of ras-p21, whic h is to induce mitogenesis in cells. A chimeric protein containing ras -p21 residues 1-65 and rap-1A residues 66-184 becomes ras-p21-like in its activity. The critical changes in sequence that result in this tra nsformation are G26N, I27H, E30D, K31E, and E45V. All of these substit utions occur in or around a critical effector domain of p21 that is in volved in interacting with GTPase activating protein (GAP), raf-p74 pr otein and inositol-3-hydroxy kinase. Using molecular dynamics, we have computed the average low energy structures for each of the three prot eins, ras-p21, rap-1A and mutant rap1A, called rap-M, that contains th ese critical amino acid substitutions. We find that rap-M more closley superimposes on ras-p21 (rms deviation 1.9 Angstrom) than on wild-typ e rap-1A (rms deviation 3.4 Angstrom). In particular, the amino termin al domains (residues 3-59) of both ras-p21 and rap-M are superimposabl e while they deviate when the average structures of these two proteins are superimposed on that of wild-type rap-1A. We have identified Pro 34 as a critical residue which may determine if the protein transforms cells or inhibits cell transformation. In addition, we have found tha t ras-p21 and rap-M proteins are superimposable in the region 96-110 e xcept at Asp 105. The 96-110 domain of ras-p21 has been found to be in volved in the binding of this protein to the nuclear transcription pro tein, jun and its kinase, jun kinase, JNK. Both segments differ in str ucture from that of the rap-1A segment at Asp 108, implicating this re sidue as also being important in determining the activity of the prote in. Overall, the oncogenic substitutions introduced into the rap-1A pr otein cause it to adopt a conformation that is very similar to that of ras-p21 rather than wild-type rap-1A.