Interaction of the N‐Terminal and C‐Terminal Domains of Elongation Factor G on Formation of Complexes with Guanyl Nucleotides

Abstract

Polarized fluorescence studies of interaction between guanyl nucleotides (GTP and GDP) and elongation factor G and its N-terminal tryptic fragment T t , carrying a fluorescent group (aminorhodamine B) at the exposed cysteine residue, has shown that binding of nucleotides by an intact EF-G molecule at neutral pH essentially affects the mobility of the fluorescent group. GTP binding changes its relaxation properties to a greater extent than GDP binding. At the same time it was demonstrated that the spectrum of relaxation times of the fluorescent group practically does not change on binding of nucleotides by the N-terminal fragment Td (in the absence of the C-terminal domain) or in the case when the three-dimensional structure of the intact EF-G molecule is destabilized (pH 10).Comparison of the relaxation properties of EF-G and its N-terminal fragment TE, carrying a fluorescent group at the exposed cysteine residue, at pH 7.5 and 10, indicates that the C-terminal domain is involved in the formation of the close environment of the exposed cysteine residue located in the N-terminal part of EF-G.A conclusion is drawn on the nucleotide-induced influence of the C-terminal domain on a change of the exposed cysteine residue environment on guanyl nucleotide binding with EF-G at neutral pH and a hypothetical model of the EF-G molecule is proposed.Elongation factor G (EF-G) catalyzes the ribosomedependent GTPase reaction [l] which proceeds through intermediate stages to the formation of an EF-G . GTP binary complex [2-61 and an EF-G . GTP . ribosome ternary complex [7 -91. In the latter, GTP is hydrolyzed and the complex dissociates into EF-G . GDP and the ribosome [2]. Thus, the change of the factor affinity to the ribosome is a result of a substitution of GTP by GDP in the binary complex. One can assume that the elongation factor G within the complexes with GTP and GDP differs in its three-dimensional structure. However, it has been shown that there is almost no difference in the circular dichroism (CD) spectra of the free elongation factor G and the factor within the complexes with GDP or GTP [lo]. Moreover, no changes in the secondary structure of EF-G on its interaction with ligands are detected even by tritium-hydrogen exchange [3]. Nonetheless, the existence of local conformational changes in the structure of EF-G on the formation of binary complexes with guanyl nucleotides has been shown by the method of hydrophobic [2] and spin-label probes [3] as well as by investigations of the rate of the exposed -SH group modification [ll,12]. If the exposed -SH group is effectively protected from modification by guanyl nucleotides [ l l , 121, then, as has been recently shown by us [13], the rate of modification of tyrosine residues in EF-G in the presence of GTP or GDP is essentially higher than in the free protein.It ha5 been reported [lo] that EF-G has a domain structure and'under the action of proteases is cleaved into a limited number of fragments resistant to further proteolysis [14,15]. The resistance of the fragments f...