p120 Ras GTPase-activating protein interacts with Ras-GTP through specific conserved residues.
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, Arg786, Lys831, and Arg925 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 Arg786, Lys831, and Arg925 residues form salt bridges with the conserved, negatively charged residues in the Ras effector loop.
Miao
W
W
Department of Medicine, Division of Hematology/Oncology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Eichelberger
L
L
Baker
L
L
Marshall
M S
MS
D000262:Adenylyl Cyclases / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D020690:GTPase-Activating Proteins; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry*; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D000262:Adenylyl Cyclases