Abstract
Members of the Ras superfamily of small GTPases (p21) are involved in the regulation of a large variety of key cellular processes, including cell differentiation and proliferation, membrane trafficking, and nuclear import and export. Based on sequence homology, this superfamily can be divided into the Ras, Rho, Ran, Arf, Rab, and Rad subfamilies, which all have distinct biological activities. All members of this superfamily act as molecular switches and become activated and capable of transducing a signal upon binding to GTP, while guanosine triphosphate (GTP) hydrolysis returns them to the inactive state. Most members of this superfamily are post-translationally modified and carry isoprenoids at their C-termini, which anchors them to the membrane.
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References
Scheffzek, K., Ahmadian, M. R., Kabsch, W., Wiesmuller, L., Lautwein, A., Schmitz, F., et al. (1997) The Ras-RasGAP complex: structural basis for GTPase activation and its loss in oncogenic Ras mutants. Science 277, 333–338.
Rittinger, K., Walker, P. A., Eccleston, J. F., Smerdon, S. J., and Gamblin, S. J.(1997) Structure at 1.65 A of RhoA and its GTPase-activating protein in complex with a transition-state analogue. Nature 389, 758–762.
Goldberg, J. (1998) Structural basis for activation of ARF GTPase: mechanisms of guanine nucleotide exchange and GTP-myristoyl switching. Cell 95, 237–248.
Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) The structural basis of the activation of Ras by Sos. Nature 394, 337–343.
Hoffman, G. R., Nassar, N., and Cerione, R. A. (2000) Structure of the Rho family GTP-binding protein Cdc42 in complex with the multifunctional regulator RhoGDI. Cell 100, 345–356.
Scheffzek, K., Stephan, I., Jensen, O. N., Illenberger, D., and Gierschik, P. (2000) The Rac-RhoGDI complex and the structural basis for the regulation of Rho proteins by RhoGDI. Nat. Struct. Biol., 7, 122–126.
Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue.Nature 375, 554–560.
Huang, L., Hofer, F., Martin, G. S., and Kim, S. H. (1998) Structural basis for the interaction of Ras with RalGDS. Nat. Struct. Biol. 5, 422–426.
Abdul-Manan, N., Aghazadeh, B., Liu, G. A., Majumdar, A., Ouerfelli, O., Siminovitch, K. A., et al. (1999) Structure of Cdc42 in complex with the GTPase-binding domain of the “Wiskott-Aldrich syndrome” protein. Nature 399, 379–383.
Mott, H. R., Owen, D., Nietlispach, D., Lowe, P. N., Manser, E., Lim, L., et al.(1999) Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK. Nature 399, 384–388.
Vetter, I. R., Nowak, C., Nishimoto, T., Kuhlmann, J., and Wittinghofer, A. (1999) Structure of a Ran-binding domain complexed with Ran bound to a GTP analogue:implications for nuclear transport. Nature 398, 39–46.
Sprang S. R. (1997) G protein mechanisms: insights from structural analysis.Annu. Rev. Biochem. 66, 639–678.
Geyer, M. and Wittinghofer, A. (1997) GEFs, GAPs, GDIs and effectors: taking a closer (3D) look at the regulation of Ras-related GTP-binding proteins. Curr.Opin. Struct. Biol. 7, 786–792.
Tucker, J., Sczakiel, G., Feuerstein, J., John, J., Goody, R. S., and Wittinghofer, A.(1986) Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site. EMBO J. 5, 1351–1358.
John, J., Frech, M., and Wittinghofer, A. (1988) Biochemical properties of Ha-ras encoded p21 mutants and mechanism of the autophosphorylation reaction. J. Biol.Chem. 263, 11,792–11,799.
Ostermeier, C. and Brunger, A. T. (1999) Structural basis of Rab effector specificity: crystal structure of the small G protein Rab3A complexed with the effector domain of rabphilin-3A. Cell 96, 363–374.
Scherer, A., John, J., Linke, R., Goody, R. S., Wittinghofer, A., Pai, R. R, et al.(1989) Crystallization and preliminary X-ray analysis of the human c-H-rasoncogene product p21 complexed with GTP analogues. J. Mol. Biol. 206, 257–259.
Graham, D. L., Eccleston, J. F., and Lowe, P. N. (1999) The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminum fluoride. Biochemistry 38, 985–991.
Guo, W., Sutcliffe, M. J., Cerione, R. A., and Oswald, R. E. (1998) Identification of the binding surface on Cdc42Hs for p21-activated kinase. Biochemistry 37, 14,030–14,037.
Leonard, D. A., Satoskar, R. S., Wu, W. J., Bagrodia, S., Cerione, R. A., and Manor, D. (1997) Use of a fluorescence spectroscopic readout to characterize the interactions of Cdc42Hs with its target/effector, mPAK-3. Biochemistry 36, 1173–1180.
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Smith, S.J.M., Rittinger, K. (2002). Preparation of GTPases for Structural and Biophysical Analysis. In: Manser, E., Leung, T. (eds) GTPase Protocols. Methods in Molecular Biology™, vol 189. Springer, Totowa, NJ. https://doi.org/10.1385/1-59259-281-3:013
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DOI: https://doi.org/10.1385/1-59259-281-3:013
Publisher Name: Springer, Totowa, NJ
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