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. 2000 Sep;9(9):1791–1800.

Interaction of mammalian mitochondrial elongation factor EF-Tu with guanine nucleotides.

Y C Cai 1, J M Bullard 1, N L Thompson 1, L L Spremulli 1
PMCID: PMC2144698  PMID: 11045624

Abstract

Elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. During the elongation cycle, EF-Tu interacts with guanine nucleotides, aa-tRNA and its nucleotide exchange factor (EF-Ts). Quantitative determination of the equilibrium dissociation constants that govern the interactions of mammalian mitochondrial EF-Tu (EF-Tu(mt)) with guanine nucleotides was the focus of the work reported here. Equilibrium dialysis with [3H]GDP was used to measure the equilibrium dissociation constant of the EF-Tu(mt) x GDP complex (K(GDP) = 1.0 +/- 0.1 microM). Competition of GTP with a fluorescent derivative of GDP (mantGDP) for binding to EF-Tu(mt) was used to measure the dissociation constant of the EF-Tu(mt) x GTP complex (K(GTP) = 18 +/- 9 microM). The analysis of these data required information on the dissociation constant of the EF-Tu(mt) x mantGDP complex (K(mGDP) = 2.0 +/- 0.5 microM), which was measured by equilibrium dialysis. Both K(GDP) and K(GTP) for EF-Tu(mt) are quite different (about two orders of magnitude higher) than the dissociation constants of the corresponding complexes formed by Escherichia coli EF-Tu. The forward and reverse rate constants for the association and dissociation of the EF-Tu(mt) x GDP complex were determined using the change in the fluorescence of mantGDP upon interaction with EF-Tu(mt). These values are in agreement with a simple equilibrium binding interaction between EF-Tu(mt) and GDP. The results obtained are discussed in terms of the recently described crystal structure of the EF-Tu(mt) x GDP complex.

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Selected References

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  1. Abel K., Yoder M. D., Hilgenfeld R., Jurnak F. An alpha to beta conformational switch in EF-Tu. Structure. 1996 Oct 15;4(10):1153–1159. doi: 10.1016/s0969-2126(96)00123-2. [DOI] [PubMed] [Google Scholar]
  2. Andersen G. R., Thirup S., Spremulli L. L., Nyborg J. High resolution crystal structure of bovine mitochondrial EF-Tu in complex with GDP. J Mol Biol. 2000 Mar 24;297(2):421–436. doi: 10.1006/jmbi.2000.3564. [DOI] [PubMed] [Google Scholar]
  3. Bullard J. M., Cai Y. C., Zhang Y., Spremulli L. L. Effects of domain exchanges between Escherichia coli and mammalian mitochondrial EF-Tu on interactions with guanine nucleotides, aminoacyl-tRNA and ribosomes. Biochim Biophys Acta. 1999 Jul 7;1446(1-2):102–114. doi: 10.1016/s0167-4781(99)00077-9. [DOI] [PubMed] [Google Scholar]
  4. Carvalho M. D., Carvalho J. F., Merrick W. C. Biological characterization of various forms of elongation factor 1 from rabbit reticulocytes. Arch Biochem Biophys. 1984 Nov 1;234(2):603–611. doi: 10.1016/0003-9861(84)90310-2. [DOI] [PubMed] [Google Scholar]
  5. Cetin R., Anborgh P. H., Cool R. H., Parmeggiani A. Functional role of the noncatalytic domains of elongation factor Tu in the interactions with ligands. Biochemistry. 1998 Jan 13;37(2):486–495. doi: 10.1021/bi970443o. [DOI] [PubMed] [Google Scholar]
  6. Crechet J. B., Parmeggiani A. Characterization of the elongation factors from calf brain. 2. Functional properties of EF-1 alpha, the action of physiological ligands and kirromycin. Eur J Biochem. 1986 Dec 15;161(3):647–653. doi: 10.1111/j.1432-1033.1986.tb10489.x. [DOI] [PubMed] [Google Scholar]
  7. Divita G., Goody R. S., Gautheron D. C., Di Pietro A. Structural mapping of catalytic site with respect to alpha-subunit and noncatalytic site in yeast mitochondrial F1-ATPase using fluorescence resonance energy transfer. J Biol Chem. 1993 Jun 25;268(18):13178–13186. [PubMed] [Google Scholar]
  8. Edmonds B. T., Bell A., Wyckoff J., Condeelis J., Leyh T. S. The effect of F-actin on the binding and hydrolysis of guanine nucleotide by Dictyostelium elongation factor 1A. J Biol Chem. 1998 Apr 24;273(17):10288–10295. doi: 10.1074/jbc.273.17.10288. [DOI] [PubMed] [Google Scholar]
  9. Giovane A., Balestrieri C., Balestrieri M. L., Servillo L. Interaction studies between elongation factor Tu and anthraniloyl-fluorescent analogues of guanyl nucleotides. Eur J Biochem. 1995 Jan 15;227(1-2):428–432. doi: 10.1111/j.1432-1033.1995.tb20405.x. [DOI] [PubMed] [Google Scholar]
  10. Hazlett T. L., Moore K. J., Lowe P. N., Jameson D. M., Eccleston J. F. Solution dynamics of p21ras proteins bound with fluorescent nucleotides: a time-resolved fluorescence study. Biochemistry. 1993 Dec 14;32(49):13575–13583. doi: 10.1021/bi00212a025. [DOI] [PubMed] [Google Scholar]
  11. John J., Rensland H., Schlichting I., Vetter I., Borasio G. D., Goody R. S., Wittinghofer A. Kinetic and structural analysis of the Mg(2+)-binding site of the guanine nucleotide-binding protein p21H-ras. J Biol Chem. 1993 Jan 15;268(2):923–929. [PubMed] [Google Scholar]
  12. John J., Sohmen R., Feuerstein J., Linke R., Wittinghofer A., Goody R. S. Kinetics of interaction of nucleotides with nucleotide-free H-ras p21. Biochemistry. 1990 Jun 26;29(25):6058–6065. doi: 10.1021/bi00477a025. [DOI] [PubMed] [Google Scholar]
  13. Kinzy T. G., Merrick W. C. Characterization of a limited trypsin digestion form of eukaryotic elongation factor 1 alpha. J Biol Chem. 1991 Mar 5;266(7):4099–4105. [PubMed] [Google Scholar]
  14. Kjeldgaard M., Nissen P., Thirup S., Nyborg J. The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation. Structure. 1993 Sep 15;1(1):35–50. doi: 10.1016/0969-2126(93)90007-4. [DOI] [PubMed] [Google Scholar]
  15. Laurberg M., Mansilla F., Clark B. F., Knudsen C. R. Investigation of functional aspects of the N-terminal region of elongation factor Tu from Escherichia coli using a protein engineering approach. J Biol Chem. 1998 Feb 20;273(8):4387–4391. doi: 10.1074/jbc.273.8.4387. [DOI] [PubMed] [Google Scholar]
  16. Leonard D. A., Evans T., Hart M., Cerione R. A., Manor D. Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy. Biochemistry. 1994 Oct 11;33(40):12323–12328. doi: 10.1021/bi00206a040. [DOI] [PubMed] [Google Scholar]
  17. Louie A., Jurnak F. Kinetic studies of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate-aminoacyl-tRNA complexes. Biochemistry. 1985 Nov 5;24(23):6433–6439. doi: 10.1021/bi00344a019. [DOI] [PubMed] [Google Scholar]
  18. Miller D. L., Weissbach H. Studies on the purification and properties of factor Tu from E. coli. Arch Biochem Biophys. 1970 Nov;141(1):26–37. doi: 10.1016/0003-9861(70)90102-5. [DOI] [PubMed] [Google Scholar]
  19. Moore K. J., Webb M. R., Eccleston J. F. Mechanism of GTP hydrolysis by p21N-ras catalyzed by GAP: studies with a fluorescent GTP analogue. Biochemistry. 1993 Jul 27;32(29):7451–7459. doi: 10.1021/bi00080a016. [DOI] [PubMed] [Google Scholar]
  20. Nagata S., Iwasaki K., Kaziro Y. Purification and properties of polypeptide chain elongation factor-1alpha from pig liver. J Biochem. 1977 Dec;82(6):1633–1646. doi: 10.1093/oxfordjournals.jbchem.a131859. [DOI] [PubMed] [Google Scholar]
  21. Nixon A. E., Brune M., Lowe P. N., Webb M. R. Kinetics of inorganic phosphate release during the interaction of p21ras with the GTPase-activating proteins, p120-GAP and neurofibromin. Biochemistry. 1995 Nov 28;34(47):15592–15598. doi: 10.1021/bi00047a026. [DOI] [PubMed] [Google Scholar]
  22. Nomanbhoy T. K., Cerione R. Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy. J Biol Chem. 1996 Apr 26;271(17):10004–10009. doi: 10.1074/jbc.271.17.10004. [DOI] [PubMed] [Google Scholar]
  23. Parmeggiani A., Swart G. W., Mortensen K. K., Jensen M., Clark B. F., Dente L., Cortese R. Properties of a genetically engineered G domain of elongation factor Tu. Proc Natl Acad Sci U S A. 1987 May;84(10):3141–3145. doi: 10.1073/pnas.84.10.3141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Polekhina G., Thirup S., Kjeldgaard M., Nissen P., Lippmann C., Nyborg J. Helix unwinding in the effector region of elongation factor EF-Tu-GDP. Structure. 1996 Oct 15;4(10):1141–1151. doi: 10.1016/s0969-2126(96)00122-0. [DOI] [PubMed] [Google Scholar]
  25. Richter M. F., Schwemmle M., Herrmann C., Wittinghofer A., Staeheli P. Interferon-induced MxA protein. GTP binding and GTP hydrolysis properties. J Biol Chem. 1995 Jun 2;270(22):13512–13517. [PubMed] [Google Scholar]
  26. Roobol K., Möller W. The role of guanine nucleotides in the interaction between aminoacyl-tRNA and elongation factor 1 of Artemia salina. Eur J Biochem. 1978 Oct 16;90(3):471–477. doi: 10.1111/j.1432-1033.1978.tb12626.x. [DOI] [PubMed] [Google Scholar]
  27. Rosenthal L. P., Bodley J. W. Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu. J Biol Chem. 1987 Aug 15;262(23):10955–10959. [PubMed] [Google Scholar]
  28. Saha S. K., Chakraburtty K. Protein synthesis in yeast. Isolation of variant forms of elongation factor 1 from the yeast Saccharomyces cerevisiae. J Biol Chem. 1986 Sep 25;261(27):12599–12603. [PubMed] [Google Scholar]
  29. Sayle R. A., Milner-White E. J. RASMOL: biomolecular graphics for all. Trends Biochem Sci. 1995 Sep;20(9):374–374. doi: 10.1016/s0968-0004(00)89080-5. [DOI] [PubMed] [Google Scholar]
  30. Schwartzbach C. J., Spremulli L. L. Bovine mitochondrial protein synthesis elongation factors. Identification and initial characterization of an elongation factor Tu-elongation factor Ts complex. J Biol Chem. 1989 Nov 15;264(32):19125–19131. [PubMed] [Google Scholar]
  31. Schwartzbach C. J., Spremulli L. L. Interaction of animal mitochondrial EF-Tu.EF-Ts with aminoacyl-tRNA, guanine nucleotides, and ribosomes. J Biol Chem. 1991 Sep 5;266(25):16324–16330. [PubMed] [Google Scholar]
  32. Song H., Parsons M. R., Rowsell S., Leonard G., Phillips S. E. Crystal structure of intact elongation factor EF-Tu from Escherichia coli in GDP conformation at 2.05 A resolution. J Mol Biol. 1999 Jan 22;285(3):1245–1256. doi: 10.1006/jmbi.1998.2387. [DOI] [PubMed] [Google Scholar]
  33. Sprinzl M. Elongation factor Tu: a regulatory GTPase with an integrated effector. Trends Biochem Sci. 1994 Jun;19(6):245–250. doi: 10.1016/0968-0004(94)90149-x. [DOI] [PubMed] [Google Scholar]
  34. Sreedharan S. P., Beck C. M., Spremulli L. L. Euglena gracilis chloroplast elongation factor Tu. Purification and initial characterization. J Biol Chem. 1985 Mar 10;260(5):3126–3131. [PubMed] [Google Scholar]
  35. Wagner A., Simon I., Sprinzl M., Goody R. S. Interaction of guanosine nucleotides and their analogs with elongation factor Tu from Thermus thermophilus. Biochemistry. 1995 Oct 3;34(39):12535–12542. doi: 10.1021/bi00039a007. [DOI] [PubMed] [Google Scholar]
  36. Watson B. S., Hazlett T. L., Eccleston J. F., Davis C., Jameson D. M., Johnson A. E. Macromolecular arrangement in the aminoacyl-tRNA.elongation factor Tu.GTP ternary complex. A fluorescence energy transfer study. Biochemistry. 1995 Jun 20;34(24):7904–7912. doi: 10.1021/bi00024a015. [DOI] [PubMed] [Google Scholar]
  37. Woriax V. L., Bullard J. M., Ma L., Yokogawa T., Spremulli L. L. Mechanistic studies of the translational elongation cycle in mammalian mitochondria. Biochim Biophys Acta. 1997 May 2;1352(1):91–101. doi: 10.1016/s0167-4781(97)00002-x. [DOI] [PubMed] [Google Scholar]
  38. Woriax V. L., Burkhart W., Spremulli L. L. Cloning, sequence analysis and expression of mammalian mitochondrial protein synthesis elongation factor Tu. Biochim Biophys Acta. 1995 Dec 27;1264(3):347–356. doi: 10.1016/0167-4781(95)00176-x. [DOI] [PubMed] [Google Scholar]
  39. Woriax V. L., Spremulli G. H., Spremulli L. L. Nucleotide and aminoacyl-tRNA specificity of the mammalian mitochondrial elongation factor EF-Tu.Ts complex. Biochim Biophys Acta. 1996 Jun 3;1307(1):66–72. doi: 10.1016/0167-4781(95)00240-5. [DOI] [PubMed] [Google Scholar]
  40. Xin H., Woriax V., Burkhart W., Spremulli L. L. Cloning and expression of mitochondrial translational elongation factor Ts from bovine and human liver. J Biol Chem. 1995 Jul 21;270(29):17243–17249. doi: 10.1074/jbc.270.29.17243. [DOI] [PubMed] [Google Scholar]
  41. Zhang Y., Li X., Spremulli L. L. Role of the conserved aspartate and phenylalanine residues in prokaryotic and mitochondrial elongation factor Ts in guanine nucleotide exchange. FEBS Lett. 1996 Aug 12;391(3):330–332. doi: 10.1016/0014-5793(96)00789-2. [DOI] [PubMed] [Google Scholar]
  42. van Damme H. T., Amons R., Möller W. Identification of the sites in the eukaryotic elongation factor 1 alpha involved in the binding of elongation factor 1 beta and aminoacyl-tRNA. Eur J Biochem. 1992 Aug 1;207(3):1025–1034. doi: 10.1111/j.1432-1033.1992.tb17139.x. [DOI] [PubMed] [Google Scholar]

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