Spoerner, Michael and Hozsa, Constantin and Poetzl, Johann A. and Reiss, Kerstin and Ganser, Petra and Geyer, Matthias and Kalbitzer, Hans Robert (2010) Conformational States of Human Rat Sarcoma (Ras) Protein Complexed with Its Natural Ligand GTP and Their Role for Effector Interaction and GTP Hydrolysis. JOURNAL OF BIOLOGICAL CHEMISTRY, 285 (51). pp. 39768-39778. ISSN 0021-9258, 1083-351X
Full text not available from this repository. (Request a copy)Abstract
The guanine nucleotide-binding protein Ras exists in solution in two different conformational states when complexed with different GTP analogs such as GppNHp or GppCH(2)p. State 1 has only a very low affinity to effectors and seems to be recognized by guanine nucleotide exchange factors, whereas state 2 represents the high affinity effector binding state. In this work we investigate Ras in complex with the physiological nucleoside triphosphate GTP. By polarization transfer P-31 NMR experiments and effector binding studies we show that Ras(wt)center dot Mg2+center dot GTP also exists in a dynamical equilibrium between the weakly populated conformational state 1 and the dominant state 2.At 278 K the equilibrium constant between state 1 and state 2 of C-terminal truncated wild-type Ras(1166) K-12 is 11.3. K-12 of full-length Ras is > 20, suggesting that the C terminus may also have a regulatory effect on the conformational equilibrium. The exchange rate (k(ex)) for Ras(wt)center dot Mg2+center dot GTP is 7 s(-1) and thus 18-fold lower compared with that found for the Ras. GppNHp complex. The intrinsic GTPase activity substantially increases after effector binding for the switch I mutants Ras(Y32F), (Y32R), (Y32W), (Y32C/C118S), (T35S), and the switch II mutant Ras(G60A) by stabilizing state 2, with the largest effect on Ras(Y32R) with a 13-fold increase compared with wild-type. In contrast, no acceleration was observed in Ras(T35A). Thus Ras in conformational state 2 has a higher affinity to effectors as well as a higher GTPase activity. These observations can be used to explain why many mutants have a low GTPase activity but are not oncogenic.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | P-31 NMR-SPECTROSCOPY; BINDING DOMAIN; H-RAS; EXCHANGE PROCESSES; CRYSTAL-STRUCTURE; STRUCTURAL BASIS; P21(RAS); P21; MOLECULES; MECHANISM; |
| 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: | 06 Jul 2020 07:19 |
| Last Modified: | 06 Jul 2020 07:19 |
| URI: | https://pred.uni-regensburg.de/id/eprint/23734 |
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