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Probing the role of loop 2 in Ras function with unnatural amino acids
H H Chung1, D R Benson, V W Cornish
1Department of Chemistry, University of California, Lawrence Berkeley Laboratory, Berkeley 94720-9989.
Abstract:
The YDPT sequence motif (residues 32-35) in loop 2 (residues 32-40) of Ha-Ras p21 protein is conserved in the Ras protein family. X-ray crystal structures have revealed significant conformational differences in this region between the GTP- and GDP-bound forms. Moreover, mutations in this region block neoplastic transformation and prevent interaction with GTPase-activating protein (GAP), suggesting that this region may contribute to the effector function of Ras. To better understand the structural features required for GAP interaction and GTPase activity, the expanded repertoire of unnatural amino acid mutagenesis has been used to investigate the roles of the key residues, Pro-34, Thr-35, and Ile-36. A Pro-34-->methanoproline mutant, in which residue 34 is locked in the trans conformation, was found to retain high levels of intrinsic and GAP-activated GTPase activity, making unlikely conformational isomerization at this position. Deletion of a single methyl group from Ile (Ile-36-->norvaline) abolished GAP activation of Ras, revealing a remarkable specificity in this protein-protein interaction. Finally, replacement of Thr-35 with diastereomeric allo-threonine led to inactivation of Ras, demonstrating the importance of the orientation of this critical residue in Ras function.
Insights
The Ras protein
Area of Science:
- Molecular Biology
- Protein Structure and Function
- Biochemistry
Background:
- The YDPT motif in Ha-Ras p21's loop 2 is conserved across the Ras family.
- Conformational changes in this loop distinguish GTP- and GDP-bound states.
- Mutations here disrupt neoplastic transformation and GTPase-activating protein (GAP) interaction.
Purpose of the Study:
- Investigate structural requirements for GAP interaction and GTPase activity.
- Elucidate the roles of Pro-34, Thr-35, and Ile-36 using unnatural amino acid mutagenesis.
Main Methods:
- Unnatural amino acid mutagenesis to probe key residues (Pro-34, Thr-35, Ile-36).
- Analysis of intrinsic and GAP-activated GTPase activity in mutant proteins.
- Structural and functional characterization of specific mutations (e.g., Pro-34 to methanoproline, Ile-36 to norvaline, Thr-35 to allo-threonine).
Main Results:
- Pro-34 mutation (locked trans conformation) retained high GTPase activity, suggesting isomerization is not critical.
- Ile-36 mutation (norvaline) abolished GAP activation, highlighting specificity in protein-protein interactions.
- Thr-35 mutation (allo-threonine) inactivated Ras, emphasizing the importance of residue orientation.
Conclusions:
- The YDPT motif's specific residues play critical roles in Ras signaling and GAP interaction.
- Ile-36 and Thr-35 are crucial for GAP binding and GTPase activation, respectively.
- Understanding these structural-functional relationships is key to Ras protein research.