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. 2014 May 16;33(10):1177-91.
doi: 10.1002/embj.201387344. Epub 2014 Mar 31.

eIF5B employs a novel domain release mechanism to catalyze ribosomal subunit joining

Bernhard Kuhle  1 , Ralf Ficner  2
Affiliations

eIF5B employs a novel domain release mechanism to catalyze ribosomal subunit joining

Bernhard Kuhle et al. EMBO J. .

Abstract

eIF5B is a eukaryal translational GTPase that catalyzes ribosomal subunit joining to form elongation-competent ribosomes. Despite its central role in protein synthesis, the mechanistic details that govern the function of eIF5B or its archaeal and bacterial (IF2) orthologs remained unclear. Here, we present six high-resolution crystal structures of eIF5B in its apo, GDP- and GTP-bound form that, together with an analysis of the thermodynamics of nucleotide binding, provide a detailed picture of the entire nucleotide cycle performed by eIF5B. Our data show that GTP binding induces significant conformational changes in the two conserved switch regions of the G domain, resulting in the reorganization of the GTPase center. These rearrangements are accompanied by the rotation of domain II relative to the G domain and release of domain III from its stable contacts with switch 2, causing an increased intrinsic flexibility in the free GTP-bound eIF5B. Based on these data, we propose a novel domain release mechanism for eIF5B/IF2 activation that explains how eIF5B and IF2 fulfill their catalytic role during ribosomal subunit joining.

Keywords: GTPase; crystal structure; molecular machines; ribosome; subunit joining; translation initiation.

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Figures

Figure 1
Figure 1. Front view of the overall structure of Chaetomium thermophilum eIF5B(517C) in the apo form
The Cα trace is shown in rainbow coloring from the N- (blue) to the C-terminus (red). The functional core of eIF5B is composed of the G domain (I) with the nucleotide binding site (arrow), domain II, domain III and domain IV.
Figure 2
Figure 2. The nucleotide-dependent conformational switch in the G domain of eIF5B
A–C Structural transition of the G domain from its apo form (A) to the GTP- (B) and the GDP-bound (C) states. P loop, switch 1 and switch 2 are colored pink in the apo state, yellow in the GTP-bound state and cyan in the GDP-bound state. Thr439, Asp476, Gly479 and His480 (Saccharomyces cerevisiae numbering) are shown as sticks; the Mg2+ ion, Na+ ion and water molecules are shown as spheres in magenta, blue and gray, respectively; nucleotides are shown as balls and sticks. D, E Network of interactions in the nucleotide binding pocket of the GTP- (D) and GDP-bound (E) factor. Direct interactions are indicated by dashed lines.
Figure 3
Figure 3. Nucleotide-dependent conformational changes in eIF5B

  1. G domain-based superposition of domains I (G), II and III from Ct-eIF5B in the apo and GTP-bound state. Both G domains are shown in gray; otherwise the same color code was used as in Fig2. The GTP-induced rearrangements result in the loss of interactions between switch 2 and helices α8 and α9 (circle) and ultimately in the release of domain III from the G domain. Domain II rotates by ∼30° relative to the G domain and is stabilized in its new orientation by the newly formed contact between β13 and β14 loop and domain I.

  2. In apo eIF5B, the inactive switch 2 (pink) forms stable contacts with helices α8 and α9 of domain III which are broken upon the GTP-induced transition of switch 2 to its active state (yellow).

  3. Comparison of the molecular switch mechanisms in EF-Tu (left) and eIF5B (right). Both trGTPases are shown in their inactive apo or GDP-bound (top) and GTP-bound (bottom) states, respectively. Functionally relevant interactions between the switch regions (yellow) of the G domain and downstream functional domains (blue) are indicated by dashed circles.

  4. The contact surface (pink) found between domain III (blue) and domains I and II in apo eIF5B (top) is entirely lost in ribosome-bound eIF5B·GDPCP (bottom; PDB: 4BVX (Fernandez et al, 2013)), where domains III and IV become stabilized between SRL and Met-tRNAiMet (not shown).

Figure 4
Figure 4. Structural model of eIF5B·GTP on the ribosome

  1. Superposition of the catalytic centers of eIF5B·GTP (yellow) and free EF-Tu·GDPNP (cyan; PDB: 1EXM). Conserved residues are shown as sticks; GDPNP is omitted for clarity.

  2. Superposition of the catalytic centers of eIF5B·GTP (yellow) and ribosome-bound EF-Tu·GDPCP (PDB: 2XQD, 2XQE) with the sarcin-ricin loop (SRL) in pink. Structural alterations relative to free eIF5B·GTP and EF-Tu·GDPNP are limited to Hiscat of EF-Tu, which is reoriented (arrow) into its active position between A2662 and Wcat.

  3. Model of domains I and II of eIF5B (orange) on the ribosome, based on the superposition with EF-Tu·GDPCP. Similar to eIF5B·GDPCP in the cryo-EM model of the 80S IC (see Supplementary Fig S5B), the G domain is associated with the SRL of the large subunit (LSU; green), while domain II interacts with the body of the small subunit (SSU; light pink).

  4. Putative interactions between the G domain and the SRL/H95 (green). Direct interactions are indicated by black dashed lines; red dashed lines indicate the positions in H95 that are cleaved by Fe(II)-BABE introduced in the position of Lys540 (Unbehaun et al, 2007). His505 lies only 3.5 Å from H95, explaining why the H505Y mutation results in a reduced affinity for the ribosome and GTPase deficiency in eIF5B (Shin et al, 2002) (see also Supplementary Table S1). The conserved Arg534 likely contributes to the interactions with H95.

Figure 5
Figure 5. eIF5B interactions with guanine nucleotides measured by ITC

  1. Heat capacity changes upon eIF5B interaction with GDP or GTP. Temperature dependency of binding enthalpy changes (ΔH) upon Ct-eIF5B(517C) interactions with GDP in the presence (•) or absence (○) of MgCl2 and of Ct-eIF5B(517C) (▾) and Ct-eIF5B(517–852) (Δ) with GTP in the presence of MgCl2. Standard deviations are given by error bars (in some cases not visible because they are smaller than the symbol size).

  2. Domains I–III of apo Ct-eIF5B. Indicated are the contact areas of domain III to domains I and II, respectively.

Figure 6
Figure 6. Schematic model of the of nucleotide cycle of eIF5B during subunit joining
eIF5B with domains I (G) to IV is shown in blue. (1) GTP binding activates eIF5B by release of domain III and rotation of domain II relative to the G domain. (2) Binding of eIF5B·GTP to the small subunit (SSU) in the absence of Met-tRNAiMet results in a non-productive complex in which eIF5B is not able to stimulate subunit joining. (3) In the correctly preassembled 48S pre-IC, the subunit-joining-competent conformation of eIF5B·GTP is stabilized by the P site-bound initiator-tRNA, (4) resulting in the recruitment of the large ribosomal subunit (LSU). (5) Formation of the 80S pre-IC triggers GTP hydrolysis in eIF5B, which reverts back into its inactive conformation, (6) followed by the dissociation of eIF5B·GDP from the elongation-competent 80S ribosome.
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