Dynamic properties of the Ras switch I region and its importance for binding to effectors.
We have investigated the dynamic properties of the switch I region of the GTP-binding protein Ras by using mutants of Thr-35, an invariant residue necessary for the switch function. Here we show that these mutants, previously used as partial loss-of-function mutations in cell-based assays, have a reduced affinity to Ras effector proteins without Thr-35 being involved in any interaction. The structure of Ras(T35S)(.)GppNHp was determined by x-ray crystallography. Whereas the overall structure is very similar to wildtype, residues from switch I are completely invisible, indicating that the effector loop region is highly mobile. (31)P-NMR data had indicated an equilibrium between two rapidly interconverting conformations, one of which (state 2) corresponds to the structure found in the complex with the effectors. (31)P-NMR spectra of Ras mutants (T35S) and (T35A) in the GppNHp form show that the equilibrium is shifted such that they occur predominantly in the nonbinding conformation (state 1). On addition of Ras effectors, Ras(T35S) but not Ras(T35A) shift to positions corresponding to the binding conformation. The structural data were correlated with kinetic experiments that show two-step binding reaction of wild-type and (T35S)Ras with effectors requires the existence of a rate-limiting isomerization step, which is not observed with T35A. The results indicate that minor changes in the switch region, such as removing the side chain methyl group of Thr-35, drastically affect dynamic behavior and, in turn, interaction with effectors. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.
Spoerner
M
M
Universität Regensburg, Institut für Biophysik und Physikalische Biochemie, Universitätsstrasse 31, 93053 Regensburg, Germany.
Herrmann
C
C
Vetter
I R
IR
Kalbitzer
H R
HR
Wittinghofer
A
A
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D018360:Crystallography, X-Ray; D006165:Guanylyl Imidodiphosphate / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D013912:Threonine; D006165:Guanylyl Imidodiphosphate; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D008274:Magnesium