Functional role of the noncatalytic domains of elongation factor Tu in the interactions with ligands

Biochemistry
R CetinA Parmeggiani

Abstract

Elongation factor (EF) Tu from Escherichia coli contains three domains, of which domain 1 (N-terminal domain) harbors the site for nucleotide binding and GTP hydrolysis. To analyze the function of domains 2 [middle (M) domain] and 3 [C-terminal (C) domain], EF-Tu(DeltaM) and EF-Tu(DeltaC) were engineered as GST-fused products and purified. Circular dichroism and thermostability showed that both constructs have conserved organized structures. Though inactive in poly(Phe) synthesis the two constructs could bind GDP and GTP with comparable micromolar affinities. Therefore, like the isolated N-terminal domain, they had lost a typical feature of EF-Tu, the >100 times stronger affinity for GDP than for GTP. EF-Tu(DeltaM) and EF-Tu(DeltaC) had an intrinsic GTPase activity comparable to that of wild-type EF-Tu. Ribosomes did not stimulate the GTPase activity of either factor, while kirromycin increased the GTPase activity of both constructs, particularly of EF-Tu(DeltaC), to a level, however, much lower than that of the intact molecule. The interaction with aa-tRNA of both mutants was >90% reduced. As a major result, their GDP-bound form could efficiently respond to EF-Ts. All four EF-Tu-specific antibiotics [kirromycin, pulvomycin, GE...Continue Reading

References

Feb 5, 1992·Journal of Molecular Biology·M Kjeldgaard, J Nyborg
Jan 1, 1985·Annual Review of Microbiology·A Parmeggiani, G W Swart
Nov 15, 1995·European Journal of Biochemistry·S NockM Sprinzl

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Citations

Dec 15, 2010·European Biophysics Journal : EBJ·Katarzyna KulczyckaJoanna Trylska
Dec 5, 1998·Biochimica Et Biophysica Acta·I M Krab, A Parmeggiani
May 16, 2002·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Hana TomincováJirí Jonák
Jan 2, 2007·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·J Jonák
May 10, 2000·The Journal of Biological Chemistry·R BinghamC Kleanthous

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