Characterization of a Ras mutant with identical GDP- and GTP-bound structures .
We previously characterized the G60A mutant of Ras and showed that the switch regions of the GTP-bound but not the GDP-bound form of this mutant adopt an "open conformation" similar to that seen in nucleotide-free Ras. Here, we mutate Lys147 of the conserved (145)SAK(147) motif in the G60A background and characterize the resulting double mutant (DM). We show that RasDM is the first structure of a Ras protein with identical GDP- and GTP-bound structures. Both structures adopt the open conformation of the active form of RasG60A. The increase in the accessible surface area of the nucleotide is consistent with a 4-fold increase in its dissociation rate. Stopped-flow experiments show no major difference in the two-step kinetics of association of GDP or GTP with the wild type, G60A, or RasDM. Addition of Sos fails to accelerate nucleotide exchange. Overexpression of the G60A or double mutant of Ras in COS-1 cells fails to activate Erk and shows a strong dominant negative effect. Our data suggest that flexibility at position 60 is required for proper Sos-catalyzed nucleotide exchange and that structural information is somehow shared among the switch regions and the different nucleotide binding motifs.
Ford
Bradley
B
Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York 11794-8661, USA.
Boykevisch
Sean
S
Zhao
Chen
C
Kunzelmann
Simone
S
Bar-Sagi
Dafna
D
Herrmann
Christian
C
Nassar
Nicolas
N
D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D018360:Crystallography, X-Ray; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D013379:Substrate Specificity; D013816:Thermodynamics; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D050505:Mutant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D018631:ras Proteins; D008239:Lysine