Conformational states of human rat sarcoma (RAS) protein complexed with its natural ligand GTP and their role for effector interaction and GTP hydrolysis
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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 (31)P NMR experiments and effector binding studies we show that Ras(wt)·Mg(2+)·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(1-166) 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)·Mg(2+)·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.
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The conformational flexibility of RAS proteins revealed by atomistic simulations is consistent with 31P-NMR spectroscopy studies.
“This conformational flexibility revealed through atomistic simulations is consistent with 31P-NMR spectroscopy studies of RAS proteins [21,52]”
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- Authors
- Spoerner, M., Hozsa, C., Poetzl, J. A., Reiss, K., Ganser, P., Geyer, M., & Kalbitzer, H. R.
- Affiliations
- University of Regensburg (DE)
- Max Planck Society (DE)
- Corresponding author
- Kalbitzer, Hans Robert
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2010-12
- Volume
- 285
- Issue
- 51
- Pages
- 39768-39778
- ISSN
- 0021-9258 (ISSN-L)
- PMC
- PMC3000958
- OpenAlex
- W2024944232
- Cited by
- 148
- References
- 43
- Citations by year
- 2026: 3 · 2025: 11 · 2024: 12 · 2023: 9 · 2022: 9 · 2021: 6 · 2020: 14 · 2019: 9 · 2018: 10 · 2017: 11 · 2016: 6 · 2015: 12 · 2014: 8 · 2013: 10 · 2012: 11
- FWCI
- 3.65
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Max Planck Institute for Plasma Physics (submittedVersion)
- University of Regensburg Publication Server (University of Regensburg) (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Receptor Mechanisms and Signaling
- Keywords
- Ras proteins, GTP binding, conformational equilibrium, effector binding, GTPase activity, guanine nucleotide exchange factor, oncogenicity, Gpp(NH)p, 31P NMR
- MeSH terms
- Animals, Guanosine Triphosphate, Humans, Proto-Oncogene Proteins p21(ras), Protein Structure, Tertiary, Nuclear Magnetic Resonance, Biomolecular, Amino Acid Substitution, Mutation, Missense, Rats
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