Spoerner, Michael and Herrmann, Christian and Vetter, Ingrid R. and Kalbitzer, Hans Robert and Wittinghofer, Alfred (2001) Dynamic properties of the Ras switch I region and its importance for binding to effectors. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 98 (9). pp. 4944-4949. ISSN 0027-8424
Full text not available from this repository. (Request a copy)Abstract
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. P-31-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 effecters. P-31-NMR spectra of pas 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 pas effecters, 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 wildtype and (T35S)Ras with effecters 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 effecters. The dynamics of the switch I region appear to be responsible for the conservation of this threonine residue in GTP-binding proteins.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | ACTIVATING PROTEIN GAP; H-RAS; CRYSTAL-STRUCTURE; DIFFRACTION DATA; P21 RAS; KINASE; DOMAIN; TRANSFORMATION; DISTINCT; COMPLEX; |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie > Prof. Dr. Dr. Hans Robert Kalbitzer |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 01 Feb 2022 16:39 |
| Last Modified: | 01 Feb 2022 16:39 |
| URI: | https://pred.uni-regensburg.de/id/eprint/41491 |
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