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X-ray crystal structures of transforming p21 ras mutants suggest a transition-state stabilization mechanism for GTP
G G Privé1, M V Milburn, L Tong
1Department of Chemistry, University of California, Berkeley 94720.
Abstract:
RAS genes isolated from human tumors often have mutations at positions corresponding to amino acid 12 or 61 of the encoded protein (p21), while retroviral ras-encoded p21 contains substitutions at both positions 12 and 59. These mutant proteins are deficient in their GTP hydrolysis activity, and this loss of activity is linked to their transforming potential. The crystal structures of the mutant proteins are presented here as either GDP-bound or GTP-analogue-bound complexes. Based on these structures, a mechanism for the p21 GTPase reaction is proposed that is consistent with the observed structural and biochemical data. The central feature of this mechanism is a specific stabilization complex formed between the Gln-61 side-chain and the pentavalent gamma-phosphate of the GTP transition state. Amino acids other than glutamine at position 61 cannot stabilize the transition state, and amino acids larger than glycine at position 12 would interfere with the transition-state complex. Thr-59 disrupts the normal position of residue 61, thus preventing its participation in the transition-state complex.
Insights
Mutant RAS proteins, common in cancer, lose GTPase activity due to specific amino acid substitutions. Structural analysis reveals how these mutations, particularly at positions 12 and 61, disrupt GTP hydrolysis, contributing to cellular transformation.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- RAS genes are frequently mutated in human tumors, leading to oncogenic p21 proteins.
- Mutations at amino acid positions 12 or 61 of p21 are common in cancer and impair GTPase activity.
- This loss of GTP hydrolysis is linked to the transforming potential of these mutant proteins.
Purpose of the Study:
- To elucidate the structural basis for the impaired GTPase activity in mutant RAS proteins.
- To propose a mechanism for the p21 GTPase reaction based on crystal structures.
- To understand how specific mutations at positions 12, 59, and 61 affect protein function.
Main Methods:
- X-ray crystallography to determine the structures of mutant p21 proteins.
- Analysis of GDP-bound and GTP-analogue-bound protein complexes.
- Biochemical data integration with structural findings.
Main Results:
- Crystal structures of mutant p21 proteins (GDP-bound and GTP-analogue-bound) were determined.
- A mechanism for the p21 GTPase reaction is proposed, highlighting the role of Gln-61.
- Mutations at position 61 prevent transition-state stabilization, and mutations at position 12 interfere with complex formation.
- Thr-59 was shown to disrupt the proper positioning of residue 61.
Conclusions:
- The Gln-61 side-chain is crucial for stabilizing the GTP transition state during hydrolysis.
- Specific mutations at positions 12 and 61 disrupt this stabilization, leading to loss of GTPase activity.
- Understanding these structural mechanisms provides insight into RAS-driven oncogenesis.