p120 Ras GTPase-activating Protein Interacts with Ras-GTP through Specific Conserved Residues

• Published in: The Journal of biological chemistry Vol. 271; no. 26; pp. 15322 - 15329
• Main Authors: Miao, W, Eichelberger, L, Baker, L, Marshall, M S
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 28.06.1996
• Subjects: Adenylyl Cyclases - chemistry; Amino Acid Sequence; Animals; Cattle; Circular Dichroism; GTPase-Activating Proteins; Humans; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Structure, Secondary; Proteins - chemistry; Proto-Oncogene Proteins p21(ras) - chemistry; ras GTPase-Activating Proteins; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Structure-Activity Relationship
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.271.26.15322

Previous structural studies of RasGAP have failed to clearly localize sites of Ras interaction to individual amino acids. Hypothesizing that sites of interaction with Ras-GTP would be conserved, 11 of the most highly conserved amino acid residues of RasGAP were changed by mutation. Each mutant protein was purified as a glutathione S -transferase catalytic domain fusion and analyzed for protein stability, Ras GTPase stimulating activity, affinity for Ras-GTP, and when possible, secondary structure. The majority of conserved positions were found to be important structurally but with no direct role in Ras interactions. However, Arg 786 , Lys 831 , and Arg 925 were observed to be essential for binding to Ras-GTP but not for protein structure. RasGAP residues 890–902 (block 3A) were observed to be homologous to residues 1540-1552 of the yeast adenylyl cyclase with amino acid substitutions in both regions resulting in increased affinity for Ras. This is the first example of a conserved Ras interaction motif in distinct Ras effector proteins. Our data are supportive of a model for GAP/Ras-GTP association in which the conserved, positively charged Arg 786 , Lys 831 , and Arg 925 residues form salt bridges with the conserved, negatively charged residues in the Ras effector loop.