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Oncogenic amino acid substitutions in the inhibitory rap-1A protein cause it to adopt a ras-p21-like conformation as
J M Chen1, P W Brandt-Rauf, M R Pincus
1Dupont Agricultural Products, Stein-Haskell Research Center, Newark, DE 19714, USA.
Abstract:
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, which 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 transformation are G26N, 127H, E30D, K31E, and E45V. All of these substitutions occur in or around a critical effector domain of p21 that is involved in interacting with GTPase activating protein (GAP), raf-p74 protein and inositol-3-hydroxy kinase. Using molecular dynamics, we have computed the average low energy structures for each of the three proteins, ras-p21, rap-1A and mutant rap1A, called rap-M, that contains these critical amino acid substitutions. We find that rap-M more closely superimposes on ras-p21 (rms deviation 1.9 A) than on wild-type rap-1A (rms deviation 3.4 A). In particular, the amino terminal domains (residues 3-59) of both RAS-p21 and rap-M are superimposable 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 that ras-p21 and rap-M proteins are superimposable in the region 96-110 except at Asp 105. The 96-110 domain of ras-p21 has been found to be involved in the binding of this protein to the nuclear transcription protein, jun and its kinase, jun kinase, JNK. Both segments differ in structure from that of the rap-1A segment at Asp 108, implicating this residue as also being important in determining the activity of the protein. Overall, the oncogenic substitutions introduced into the rap-1A protein cause it to adopt a conformation that is very similar to that of ras-p21 rather than wild-type rap-1A.
Insights
Mutating the rap-1A protein with ras-p21 sequences changes its function to promote cell growth. Molecular dynamics reveal these mutations cause rap-1A to adopt a ras-p21-like structure, particularly in key domains.
Area of Science:
- Molecular Biology
- Biochemistry
- Structural Biology
Background:
- Rap-1A is a G-protein that inhibits ras-p21-induced mitogenesis.
- Ras-p21 is a G-protein that promotes cell proliferation.
Purpose of the Study:
- To investigate the structural and functional consequences of introducing ras-p21 mutations into rap-1A.
- To identify key residues responsible for the altered activity of the chimeric protein.
Main Methods:
- Construction of a chimeric protein (rap-M) with ras-p21 residues in rap-1A.
- Molecular dynamics simulations to compute average low-energy structures.
- Structural superposition and root-mean-square deviation (RMSD) analysis.
Main Results:
- Rap-M adopts a structure more similar to ras-p21 (RMSD 1.9 Å) than wild-type rap-1A (RMSD 3.4 Å).
- Amino-terminal domains (residues 3-59) of ras-p21 and rap-M are superimposable.
- Differences in the 96-110 region, particularly at Asp 105 (rap-M) vs. Asp 108 (rap-1A), are noted.
Conclusions:
- Oncogenic substitutions in rap-1A induce a conformation resembling ras-p21.
- Pro 34 and Asp 105/108 are critical residues influencing protein activity (transformation vs. inhibition).
- The structural changes explain the functional shift from inhibition to promotion of mitogenesis.