Structure of the archaeal translation initiation factor aIF2 beta from Methanobacterium thermoautotrophicum: implications for translation initiation

Abstract

aIF2 beta is the archaeal homolog of eIF2 beta, a member of the eIF2 heterotrimeric complex, implicated in the delivery of Met-tRNA(i)(Met) to the 40S ribosomal subunit. We have determined the solution structure of the intact beta-subunit of aIF2 from Methanobacterium thermoautotrophicum. aIF2 beta is composed of an unfolded N terminus, a mixed alpha/beta core domain and a C-terminal zinc finger. NMR data shows the two folded domains display restricted mobility with respect to each other. Analysis of the aIF2 gamma structure docked to tRNA allowed the identification of a putative binding site for the beta-subunit in the ternary translation complex. Based on structural similarity and biochemical data, a role for the different secondary structure elements is suggested.

Figure 1.

Sequence alignment of amino acid sequences of archaeal IF2β: M. thermoautotrophicum (Mt_aIF2β), M. jannaschii (Mj_aIF2β), P. abyssi (Pa_aIF2β); eukaryotic IF2β: Saccharomyces cerevisiae (Sc_eIF2β), Homo sapiens (Hs_eIF2β), Caenorhabditis elegans (Ce_eIF2β); and N-terminal eIF5 from H. sapiens (Hs_eIF5) and C. elegans (Ce_eIF5). Secondary structure elements are shown on top. Helix α1 is hypothetical and is proposed based on secondary structure predictions and chemical shift index. Swissprot ID numbers for the sequences shown are O27797, Q57562, O58312, PO9064, P20042, Q21230, P55010, and Q22918.

Figure 2.

Plots of ¹H-¹⁵N heteronuclear NOE, number of NOE constraints and RMSD statistics for Mt_aIF2β. (A) ¹H-¹⁵N heteronuclear NOE values obtained at a proton frequency of 500 MHz. (B) A summary of all unambiguous NOEs: intraresidue, sequential, medium, and long-range NOEs are shown in dark gray, black, light gray, and white, respectively. (C) A comparison of the average backbone RMSD (Å) per residue of the 20 lowest energy structures calculated without (diamonds) and with (circles) ¹H-¹⁵N residual dipolar couplings. The fit was calculated for residues 30 to 130.

Figure 3.

Stereo drawing of the superposition for backbone atoms (residues 30–130) of 10 low energy structures for Mt_aIF2β. The RMSD to the mean structure for backbone atoms in the folded region (30–130) is 0.76 ± 0.12 Å. RMSD values for the core domain (30–98) and the zinc finger (98–130) are 0.54 ± 0.11 and 0.63 ± 0.16 Å, respectively.

Figure 4.

Structure of Mt_aIF2β. (A) Ribbon representation of Mt_aIF2β, excluding the unfolded N terminus. The order of the β strands and α helices is indicated, and the sphere represents the zinc ion. (B) Topology diagram of Mt_aIF2β showing the conectivity between the secondary structure elements. β-Strands and α-helices are colored purple and greens, respectively. Ribbon diagram was generated using MOLSCRIPT (Kraulis 1991).

Figure 5.

Mapping of different biochemical data to the Mt_aIF2β structure. (A) Structurally related ELK-1, GABP, and SAP-1 are shown with their cognate nucleic acid, suggesting a role for the HTH motif of aIF2β in nucleic acid recognition. Cartoons were made with MOLSCRIPT (Kraulis 1991). (B) Electrostatic surface plot of Mt_aIF2β revealing potential RNA binding regions. Acidic and basic residues are colored red and blue, respectively. Surface generated with MOLMOL (Koradi et al. 1996). Residues 1–28 were excluded for clarity. (C) Biochemical data for a/eIF2β mapped to a model of P. abyssi aIF2γ (green, PDB identifier 1KK1) bound to tRNA (magenta, PDB identifier 1B23). A location for aIF2β in the ternary complex is suggested. Regions in aIF2β involved in GTP, tRNA_i, and aIF2γ interactions are circled and connected to their corresponding ligand (see text for details). Positions that differentiate initiator tRNA from elongator tRNA are colored in blue. aIF2β has been colored based on sequence conservation where red represents highly conserved residues.