Mapping the Effector Region in Thermus thermophilus Elongation Factor Tu

Marcus E. Peter, Norbert K. Schirmer, Christian O A Reiser, Mathias Sprinzl*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

Native elongation factor Tu from Thermus thermophilus is initially attacked by various en-doproteases in a region spanning amino acid residues 40–70. By comparing the hydrolysis rates of nucleotide-free and GDP-bound EF-Tu, only a small difference was observed for the tryptic cleavage at Arg-59. Protease V-8 attacks Glu-55 only in a GDP/GTP form, whereas this enzyme exclusively hydrolyze Asn-64 in nucleotide-free EF-Tu, even when the protein had been previously cleaved at Arg-59. Binding of GDP leads to a 42-fold decreased rate of hydrolysis by the Lys-C protease at Lys-52. It also reduces the accessibility of Lys-275 to trypsin, reflecting a “long-range” effect from nucleotide binding domain I to domain II. Only slight differences were observed in the rate of hydrolysis at all positions in the GDP- versus the GTP-bound form. The intrinsic GTPase activity was slightly reduced in trypsin-treated EF-Tu, significantly impaired in EF-Tu cleaved at Lys-52, and completely abolished in EF-Tu cleaved at Asn-64. No ribosome-induced GTPase activity was observed for protease-cleaved EF-Tu's. Treatment of these proteins with periodate-oxidized GDP or GTP followed by cyanoborohydride led to covalent modification of the new N-terminus located exclusively within region 52–60. The highest reactivity was shown by the N-terminus of Glu-56. Additionally, lysine residues in the native protein sensitive to affinity labeling [Peter, M. E., Wittmann-Liebold, B., & Sprinzl, M. (1988) Biochemistry 27, 9132–9139] lost their reactivity upon cleavage of EF-Tu in region 52–60, suggesting an altered structure of the cleaved protein. We conclude that region 52–60 is modulating the nucleotide binding site structure and is involved in the intramolecular signal transduction for the GTPase activation.

Original languageEnglish (US)
Pages (from-to)2876-2884
Number of pages9
JournalBiochemistry
Volume29
Issue number11
DOIs
StatePublished - Mar 1 1990

ASJC Scopus subject areas

  • Biochemistry

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