Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes

Abstract

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.

Figure 1.

Overall structure of Oryctolagus cuniculus eEF1A2*GDP. (A) eEF1A2 is crystallized as a dimer. Three domains of eEF1A2 are colored as follows: domain I in yellow, domain II in green and domain III in blue. The Switch I and II regions are designated as S-I and S-II. The GDP is shown as a ball-and-stick representation. The N-terminus and the C-terminus are marked as Nt and Ct, respectively. (B) Presentation of the helices and beta-folds in domain I of eEF1A2*GDP. The α-helices are labeled by upper case letters, and β-strands are labeled by lower case letters. (C) Location of phosphorylated Thr239 and Ser163 in eEF1A2. (D) Network of interactions in the nucleotide-binding pocket of the GDP-bound eEF1A2. Magnesium ion is not shown for sake of clarity. (E) Electron density map corresponding to the molecule of GDP bound to eEF1A. Magnesium ion is colored in green.

Figure 2.

Main conformational rearrangements upon GTP hydrolysis. (A and B) A′ and A* helical arrangement in the mammalian and archaeal elongation factors. eEF1A2*GDP is superimposed with aEF1A*GTP (A) and aEF1A*GDP (B). The structure of eEF1A2 is colored in red with GDP in dark gray, aFF1A*GDP and aEF1A*GTP are colored blue with GDP or GTP in light gray. Mg²⁺ ions are colored light green in eEF1A2*GDP and dark green in aEF1A*GDP or aEF1A*GTP. Note the unwinding of helix A′ in aEF1A*GTP and similar orientation of helices A* in eEF1A2*GDP and aEF1A*GTP. (C) Tyr56 and Trp58 are responsible for an interaction of the A′ and A helices. Mutation of Tyr56 or Trp58 impairs the A′- A interaction during GTP binding and hydrolysis. (D) Superimposition of the domain II+III units of eEF1A2*GDP (pink), eEF1A_y (green) in the complex with eEF1Bα (not shown for sake of clarity) and aEF1A*GTP (blue) after alignment of domain I (not shown for sake of clarity) of all complexes. (E) Superimposition of the domain II+III units of bacterial EF-Tu in GDP (pink), GEF-induced (green) and GTP (blue) conformations after alignment of domain I (not shown for sake of clarity) of all complexes.

Figure 3.

Mg²⁺ does not influence nucleotide exchange in eEF1A2. Mg²⁺ (black) and EDTA (red) do not have an impact on spontaneous (A) and eEF1Bα-catalyzed (B) nucleotide exchange process. The eEF1A2 concentration in the incubation mixture was 692 nM and eEF1Bα - 4 nM. The concentration of either Mg²⁺ or EDTA was 10 mM. Goodness (R²) of single exponential fits was calculated to be >0.999 for nucleotide exchange in the presence of both Mg²⁺ and EDTA. (C) Mg²⁺ contributes to GDP binding in EF-Tu rather than in eEF1A2.

Figure 4.

Mechanism of GEF-induced nucleotide exchange in eEF1A. (A) Conformation of the Switch I–Switch II region in eEF1A*GDP and in eEF1A_y-GEF. (B) Conformation of the GDP binding site in the absence and in the presence of GEF. Note the 180° rotation of Asp91. K205 and Q204 of eEF1Bα (numeration of yeast eEF1Bα) are shown in light brown. (C) Arrangement of the GDP-bound structures of eEF1A and EF-Tu. (D) Changes introduced in the GDP-bound structures of eEF1A and EF-Tu by corresponding GEFs. Eukaryotic GEF eEF1Bα directly disrupts contact of Ser21 with the β-phosphate, induces the conformational switch in P-loop leading to the disruption of Asp17 contact and prompts the conformational change in Switch II inducing 180° rotation of Asp91, with subsequent disruption of the Lys20 contact with the β-phosphate. Note the non-involvement of Mg²⁺ and GEF-induced rotation of Asp91 by 180°. Prokaryotic GEF EF-Ts causes conformational change in P loop, precluding contacts of Trp25 with Mg²⁺ linked to the β-phosphate, inducing rotation of Asp21 away from the β-phosphate and switch of Lys24 toward Asp81. Note the direct role of Mg²⁺ in the GDP stabilization, as well as similar positions of Asp81 in the GDP- and GEF-bound conformations. Water molecules are shown in blue. Magnesium ion is shown in gray. Compounds which are invisible in the structure, depicted as semi-transparent.