Kiel, Christina and Filchtinski, Daniel and Spoerner, Michael and Schreiber, Gideon and Kalbitzer, Hans Robert and Herrmann, Christian (2009) Improved Binding of Raf to Ras.GDP Is Correlated with Biological Activity. JOURNAL OF BIOLOGICAL CHEMISTRY, 284 (46). pp. 31893-31902. ISSN 0021-9258, 1083-351X
Full text not available from this repository. (Request a copy)Abstract
The GTP-binding protein Ras plays a central role in the regulation of various cellular processes, acting as a molecular switch that triggers signaling cascades. Only Ras bound to GTP is able to interact strongly with effector proteins like Raf kinase, phosphatidylinositol 3-kinase, and RalGDS, whereas in the GDP-bound state, the stability of the complex is strongly decreased, and signaling is interrupted. To determine whether this process is only controlled by the stability of the complex, we used computer-aided protein design to improve the interaction between Ras and effector. We challenged the Ras.Raf complex in this study because Raf among all effectors shows the highest Ras affinity and the fastest association kinetics. The proposed mutations were characterized as to their changes in dynamics and binding strength. We demonstrate that Ras-Raf interaction can only be improved at the cost of a loss in specificity of Ras.GTP versus Ras.GDP. As shown by NMR spectroscopy, the Raf mutation A85K leads to a shift of Ras switch I in the GTP-bound as well as in the GDP-bound state, thereby increasing the complex stability. In a luciferase-based reporter gene assay, Raf A85K is associated with higher signaling activity, which appears to be a mere matter of Ras-Raf affinity.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PROTEIN-PROTEIN ASSOCIATION; P-31 NMR-SPECTROSCOPY; DISSOCIATION STIMULATOR; EFFECTOR SPECIFICITY; BIOCHEMICAL-ANALYSIS; STRUCTURAL BASIS; H-RAS; R-RAS; COMPLEX; DOMAIN; |
| 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: | 02 Sep 2020 05:35 |
| Last Modified: | 02 Sep 2020 05:35 |
| URI: | https://pred.uni-regensburg.de/id/eprint/28136 |
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