NMR characterization of full-length farnesylated and non-farnesylated H-Ras and its implications for Raf activation
- PMID: 15491620
- DOI: 10.1016/j.jmb.2004.08.106
NMR characterization of full-length farnesylated and non-farnesylated H-Ras and its implications for Raf activation
Abstract
The C terminus, also known as the hypervariable region (residues 166-189), of H-, N-, and K-Ras proteins has sequence determinants necessary for full activation of downstream effectors such as Raf kinase and PI-3 kinase as well as for the correct targeting of Ras proteins to lipid rafts and non-raft membranes. There is considerable interest in understanding how residues in the extreme C terminus of the different Ras proteins and farnesylation of the CaaX box cysteine affect Ras membrane localization and allosteric activation of Raf kinase. To provide insights into the structural and dynamic changes that occur in Ras upon farnesylation, we have used NMR spectroscopy to compare the properties of truncated H-Ras (1-166), to non-processed full-length H-Ras (residues 1-185) and full-length (1-189) farnesylated H-Ras. We report that the C-terminal helix alpha-5 extends to residue N172, and the remaining 17 amino acid residues in the C terminus are conformationally averaged in solution. Removal of either 23 or 18 amino acid residues from the C terminus of full length H-Ras generates truncated H-Ras (1-166) and H-Ras (1-171) proteins, respectively, that have been structurally characterized and are biochemical active. Here we report that C-terminal truncation of H-Ras results in minor structural and dynamic perturbations that are propagated throughout the H-Ras protein including increased flexibility of the central beta-sheet and the C-terminal helix alpha-5. Ordering of residues in loop-2, which is involved in Raf CRD binding is also observed. Farnesylation of full-length H-Ras at C186 does not result in detectable conformational changes in H-Ras. Chemical shift mapping studies of farnesylated and non-farnesylated forms of H-Ras with the Raf-CRD show that the farnesyl moiety, the extreme H-Ras C terminus and residues 23-30, contribute to H-Ras:Raf-CRD interactions, thereby increasing the affinity of H-Ras for the Raf-CRD.
Similar articles
-
B- and C-RAF display essential differences in their binding to Ras: the isotype-specific N terminus of B-RAF facilitates Ras binding.J Biol Chem. 2007 Sep 7;282(36):26503-16. doi: 10.1074/jbc.M607458200. Epub 2007 Jul 16. J Biol Chem. 2007. PMID: 17635919
-
Structural basis for intramolecular interaction of post-translationally modified H-Ras•GTP prepared by protein ligation.FEBS Lett. 2017 Aug;591(16):2470-2481. doi: 10.1002/1873-3468.12759. Epub 2017 Aug 2. FEBS Lett. 2017. PMID: 28730604
-
Prenylation is not necessary for endogenous Ras activation in non-malignant cells.J Cell Biochem. 2006 Feb 1;97(2):412-22. doi: 10.1002/jcb.20641. J Cell Biochem. 2006. PMID: 16187291
-
Interactions between Ras and Raf: key regulatory proteins in cellular transformation.Mol Reprod Dev. 1995 Dec;42(4):493-9. doi: 10.1002/mrd.1080420418. Mol Reprod Dev. 1995. PMID: 8607981 Review.
-
Nonconventional trafficking of Ras associated with Ras signal organization.Traffic. 2006 Sep;7(9):119-26. doi: 10.1111/j.1600-0854.2006.00459.x. Traffic. 2006. PMID: 16824054 Review.
Cited by
-
Conformational and Dynamical Effects of Tyr32 Phosphorylation in K-Ras: Molecular Dynamics Simulation and Markov State Models Analysis.J Phys Chem B. 2019 Sep 12;123(36):7667-7675. doi: 10.1021/acs.jpcb.9b05768. Epub 2019 Aug 30. J Phys Chem B. 2019. PMID: 31419909 Free PMC article.
-
Transformation efficiency of RasQ61 mutants linked to structural features of the switch regions in the presence of Raf.Structure. 2007 Dec;15(12):1618-29. doi: 10.1016/j.str.2007.10.011. Structure. 2007. PMID: 18073111 Free PMC article.
-
Functional specificity of ras isoforms: so similar but so different.Genes Cancer. 2011 Mar;2(3):216-31. doi: 10.1177/1947601911408081. Genes Cancer. 2011. PMID: 21779495 Free PMC article.
-
Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain.Sci Rep. 2018 May 31;8(1):8461. doi: 10.1038/s41598-018-26832-4. Sci Rep. 2018. PMID: 29855542 Free PMC article.
-
The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways.PLoS One. 2011 Jan 6;6(1):e15866. doi: 10.1371/journal.pone.0015866. PLoS One. 2011. PMID: 21253588 Free PMC article.
Publication types
MeSH terms
Substances
Grants and funding
LinkOut - more resources
Full Text Sources
Other Literature Sources
Research Materials
Miscellaneous