Dynamic properties of the Ras switch I region and its importance for binding to effectors
10.1073/pnas.081441398
Crossref journal-article
Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences (341)
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. 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.

Bibliography

Spoerner, M., Herrmann, C., Vetter, I. R., Kalbitzer, H. R., & Wittinghofer, A. (2001). Dynamic properties of the Ras switch I region and its importance for binding to effectors. Proceedings of the National Academy of Sciences, 98(9), 4944–4949.

Authors 5
  1. Michael Spoerner (first)
  2. Christian Herrmann (additional)
  3. Ingrid R. Vetter (additional)
  4. Hans Robert Kalbitzer (additional)
  5. Alfred Wittinghofer (additional)
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Dates
Type When
Created 23 years, 1 month ago (July 26, 2002, 10:44 a.m.)
Deposited 3 years, 2 months ago (June 7, 2022, 1:51 a.m.)
Indexed 2 days, 1 hour ago (Aug. 29, 2025, 6:11 a.m.)
Issued 24 years, 4 months ago (April 24, 2001)
Published 24 years, 4 months ago (April 24, 2001)
Published Online 24 years, 4 months ago (April 24, 2001)
Published Print 24 years, 4 months ago (April 24, 2001)
Funders 0

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@article{Spoerner_2001, title={Dynamic properties of the Ras switch I region and its importance for binding to effectors}, volume={98}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.081441398}, DOI={10.1073/pnas.081441398}, number={9}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Spoerner, Michael and Herrmann, Christian and Vetter, Ingrid R. and Kalbitzer, Hans Robert and Wittinghofer, Alfred}, year={2001}, month=apr, pages={4944–4949} }