Structure of a transient intermediate for GTP hydrolysis by ras.
The flexibility of the conserved 57DTAGQ61 motif is essential for Ras proper cycling in response to growth factors. Here, we increase the flexibility of the 57DTAGQ61 motif by mutating Gln61 to Gly. The crystal structure of the RasQ61G mutant reveals a new conformation of switch 2 that bears remarkable structural homology to an intermediate for GTP hydrolysis revealed by targeted molecular dynamics simulations. The mutation increased retention of GTP and inhibited Ras binding to the catalytic site, but not to the distal site of Sos. Most importantly, the thermodynamics of RafRBD binding to Ras are altered even though the structure of switch 1 is not affected by the mutation. Our results suggest that interplay and transmission of structural information between the switch regions are important factors for Ras function. They propose that initiation of GTP hydrolysis sets off the separation of the Ras/effector complex even before the GDP conformation is reached.
Department of Physiology and Biophysics, Basic Sciences Tower, Stony Brook University, Stony Brook, New York 11794, USA.
Bradley
B
Ford
Viktor
V
Hornak
Holly
H
Kleinman
Nicolas
N
Nassar
D018360:Crystallography, X-Ray; D005954:Glucosephosphate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry; D015689:Oncogene Protein p21(ras) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013014:SOS Response, Genetics / Q000235:genetics; D015398:Signal Transduction; D048490:raf Kinases / Q000737:chemistry / Q000378:metabolism
D050505:Mutant Proteins; D009711:Nucleotides; D006160:Guanosine Triphosphate; D005954:Glucosephosphate Dehydrogenase; D048490:raf Kinases; D015689:Oncogene Protein p21(ras); D008274:Magnesium