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from Methanobacterium thermoautotrophicum: Implications for translation initiation
1 McGill University, Department of Biochemistry, McIntyre Medical Science Building, Montréal, Québec H3G 1Y6, Canada
2 Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada
3 Ontario Cancer Institute, Toronto, Ontario M5G 2M9, Canada
4 Montreal Joint Centre for Structural Biology, Montréal, Québec, Canada
Reprint requests to: Kalle Gehring, McGill University, Department of Biochemistry, McIntyre Medical Science Building, 3655 Promenade Sir William Osler, Montréal, Québec H3G 1Y6, Canada; e-mail: Kalle. Gehring{at}mcgill.ca; fax: (514) 398-7384.
(RECEIVED November 4, 2003; FINAL REVISION December 1, 2003; ACCEPTED December 1, 2003)
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Abstract |
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is the archaeal homolog of eIF2, a member of the eIF2 heterotrimeric complex, implicated in the delivery of Met-tRNAiMet to the 40S ribosomal subunit. We have determined the solution structure of the intact -subunit of aIF2 from Methanobacterium thermoautotrophicum. aIF2 is composed of an unfolded N terminus, a mixed 
/ 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
structure docked to tRNA allowed the identification of a putative binding site for the -subunit in the ternary translation complex. Based on structural similarity and biochemical data, a role for the different secondary structure elements is suggested.
Keywords: aIF2; translation initiation; archaebacteria; NMR
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03506604.
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Introduction |
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, , and subunits, with high sequence similarity to their eukaryotic counterparts (eIF2). eIF2 plays a critical role in the initiation of protein synthesis by forming a ternary complex with GTP and the aminoacylated initiator methionyl-tRNA (Met-tRNAiMet). This complex binds to the small ribosomal subunit (Bell and Jackson 1998), and with the aid of other translation factors scans from the 5' end of mRNA. Upon recognition of the initiation codon, GTP is hydrolyzed and the eIF2-GDP complex is released. This leads to assembly of the 80S ribosome at the initation codon and the start of protein elongation. The recycling of eIF2 between successive rounds of translation requires an additional protein factor, the guanine nucleotide exchange factor IF2B, which catalyzes the exchange of GDP bound to eIF2 for GTP (Kimball 1999; Pestova and Hellen 2000).
Distinct functions have been observed for each subunit of eIF2. The subunit is a global regulator of protein synthesis in eukaryotes. Phosphorylation of eIF2 regulates the exchange rate of GDP to GTP in a/eIF2, altering its availability for translation initiation througn the inhibition of Met-tRNAiMet binding (Pain 1996). The subunit is responsible for GTP binding, and its similarity to EF-Tu (~27% identity, ~50% similarity) allowed the identification of the Met-tRNAiMet binding region (Schmitt et al. 2002). The subunit of eIF2 is implicated in a variety of interactions with other translation factors. For example, its N terminus binds to eIF5, the GTPase activating factor (GAP) for eIF2, and to the 
subunit of the exchange factor eIF2B (Asano et al. 1999). This region has also been shown to bind RNA in vitro through three lysine repeats (Laurino et al. 1999). The C-terminal region of eIF2 contains another potential RNA binding motif. Mutations in this C2–C2 zinc finger result in spontaneous GTPase activity and alter the correct recognition of the AUG codon (Huang et al. 1997). The subunit has also been implicated in binding to the 
subunit of eIF2B and to crosslink GTP and Met-tRNAiMet (Bommer and Kurzchalia 1989; Gaspar et al. 1994). Archaeal aIF2 has ~50% similarity and ~30% identity to the C-terminal half of eukaryotic IF2 but lacks of N-terminal polysine tracts, which are ubiquitous in eukaryotes (Laurino et al. 1999; Thompson et al. 2000). a/eIF2 also shares a high degree of sequence similarity with eIF5, an eukaryotic initiation factor important for stimulating hydrolysis of GTP by the ternary complex.
Solution structures of the N- and C-terminal domains of aIF2 from Methanococcus jannaschii (Mj_aIF2) have been recently determined (Cho and Hoffman 2002). In this article, we present the solution structure of the intact archaeal translation initiation factor 2 from Methanobacterium thermoautotrophicum, including the interdomain linker, absent in the Mj_aIF2 structure. Additionally, based on comparison with structuraly similar proteins and previous known biochemical data, we propose roles for the different regions of a/eIF2 in translation initiation.
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was determined by heteronuclear multidimensional NMR spectroscopy and calculated using standard molecular dynamics protocols. The protein used for structural studies included all 135 residues from Mt_aIF2 and an additional three residues from the purification tag. Backbone, 1H, 15N, and 13C assignments for all residues (excluding K29 and F86) and >95% of the side-chain protons were obtained. Regular secondary structure was determined from the chemical shift index (Wishart and Sykes 1994) and confirmed by observation of characteristic NOEs. The position of secondary structure elements relative to the sequence is shown on Figure 1
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). {1H}-15N NOEs indicate that under our conditions residues 1–30 are unfolded (Fig. 2). The Mt_aIF2 structure can be divided in three regions: an unfolded N terminus, a core domain, and a C-terminal zinc-finger domain. The core and zinc-finger domains have a backbone RMSDs to the mean of 0.54 Å and 0.63 Å, respectively (Table 1). The zinc finger is mobile with respect to the core domain, as evidenced by the lack of long-range NOEs between these two regions. An average orientation of both domains was obtained by the use of residual dipolar couplings, giving a backbone RMSD to the mean of 0.76 Å for residues 30 to 130 (Fig. 3).
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-helices (2, 3, and 4) packed against an antiparallel four-stranded -sheet (1, 2, 3, and 4), in topology (Fig. 4). The -sheet region is formed by residues 32–35, 38–41, 71–73, and 77–80, and the helical bundle comprises residues 44–51, 55–64, and 87–98. The zinc-finger domain (residues 99–135) is composed of three antiparallel -strands (5, 6, and 7) encompasing residues 112–116, 120–124, and 128–130, respectively. Zinc is required for the structural stability of this domain (Gutierrez et al. 2002), and is coordinated by four cysteines at positions 102, 105, 123, and 126. Helix 4, links the core domain and the zinc finger. The absence of significant chemical shift changes in the core domain upon folding of the C terminus (Cho and Hoffman 2002; Gutierrez et al. 2002), the lack of NOEs between the two domains and their different RDC alignment tensors suggest that there are minimal interactions between the core domain and the zinc finger. Superposition of the core and zinc finger domains of Mj_aIF2 to Mt_aIF2 gives backbone RSMDs of 2.24 Å. The most divergent parts are 3, 4, and the loop connecting 4 and 5. Only the coordinates for the separate domains of Mj_aIF2 are available.
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(Fig. 2
; data not shown). The unstructured N terminus exhibits intermediate NOE values for residues in belonging to helix 1 consistent with the presence of a partially populated helix in this region (Eliezer et al. 2000). The structured core and zinc finger domains exhibit a trimmed weighted correlation time of ~8.45 nsec, which is in excellent agreement with the predicted value for a 135 residue protein using the Stokes-Einstein equation (8.5 nsec at 303 K), indicating that these domains do not tumble completely freely of each other. However, the lower-than-expected NOE value in the structured domains (trimmed mean 0.73) indicates that some mobility on the picosecond–nanosecond time scales is present. This was confirmed by the poor fits of the R2/R1 ratios to nonisotropic rotational diffusion models (Lee et al. 1997; Osborne and Wright 2001), despite the high degree of anisotropy predicted from hydrodynamics calculations (D||/D
= 1.43). A possible source for these motions can be restricted interdomain motions, which would be consistent with the lack of observable NOE contacts between the domains and the large RMSD prior to refinement with residual dipolar couplings.
Comparative analysis of the Mt_aIF2 structure
The structural classification databases Dali/FSSP (Holm and Sander 1998) and SCOP (Hubbard et al. 1997) were used for comparative analysis of the Mt_aIF2 structure. The coordinates were compared with known structures using the Dali and SSM search tools. Fifteen proteins with Z scores higher than 2.0 showed similarity to either the core domain or the zinc finger (Table 2). All the structures related to the core domain are nucleic acid binding proteins with a helix-turn-helix (HTH) structural motif. This motif is composed of an helix, a linking or turn region, and a second helix (recognition helix) involved in sequence specific nucleic acid interactions. The most closely related structures are Elk-1 (Mo et al. 2000), heat-shock transcription factor (Damberger et al. 1994), GABP (Batchelor et al. 1998), SAP-1 (Mo et al. 1998), PU.1 (Pio et al. 1996), Mu repressor (Wojciak et al. 2001), Mu transposase (Clubb et al. 1994), Ribosomal protein L11 (Markus et al. 1997), and hRFX1 (Gajiwala et al. 2000). Six of these proteins have been solved with their cognate DNA. This interesting result suggests a role for 2, 3, 3, and 4 aIF2 in binding to nucleic acids (Fig. 5A).
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is involved in the recognition of nucleic acids. |
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in translation initiation can be deduced based on our structure and established biochemical facts. The central portion of eukaryotic IF2 (equivalent to the unfolded N terminus of Mt_aIF2) is necessary for the interaction with eIF2 as shown by immunoprecipitation, yeast two-hybrid, and GST pull-down assays (Thompson et al. 2000; Hashimoto et al. 2002). C, C, and H secondary chemical shifts suggest the presence of a partially folded helix (1) at the N terminus (Gutierrez et al. 2002). Secondary structure predictions show that this region could form a highly amphipathic helix that could interact with a hydrophobic patch on aIF2. Mutations in this region of aIF2 affect the hydrolysis of GTP by the subunit (Hashimoto et al. 2002). Surface potential analysis of the aIF2 structure reveals a conserved hydrophobic patch formed by 6 and 6 (residues 175–179 and 188–197). The loop connecting these elements is involved in GTP binding. This region could constitute a binding site for the N terminus of aIF2.
Analysis of the surface potential of aIF2 reveals the presence of clustered basic residues typical of RNA binding proteins. Figure 5B shows the surface potential, as calculated with the program MOLMOL (Koradi et al. 1996). Negative patches originate from the charges on residues E46, D49, E65, E73, E90, E93, D94, E104, D109, and E115. The observed distribution of basic residues suggests a putative interaction site comprised by R53, H57, K60, R64, and R76 in helices 2, 3, and strands 3 and 4. R76 and H57 are conserved throughout most of the a/eIF2 sequences, and R53 is completely conserved. The closely related structures obtained from the DALI search suggest a RNA binding region in the core domain of aIF2. The key residue for this function is probably R53, located in the loop connecting 2 and 3. From the sequence alignments, it seems that at least another basic residue has to be present in helix 3 at positions equivalent to 57, 60, or 64 of Mt_aIF2. Unfortunately, no functional studies of mutants in this region have been done.
Several mutagenesis studies have shown the importance of C-terminal residues for the function of eIF2 in yeast (Donahue et al. 1988; Castilho-Valavicius et al. 1992). Of particular interest are the nonconservative substitutions R248T, R253I, R253S, L254P, V268F, and V268G (corresponding to positions R114, R117, I118, and L132 of the archaeal protein) that are located at the tip of the zinc finger and allow translation to initiate at UUG codons instead of AUG. As zinc-finger domains are normally associated with the recognition of sequence specific double-stranded nucleic acids, the C terminus of aIF2 is likely to constitute a second RNA binding region.
Based on the aIF2 structure from Pyrococcus abyssi docked to tRNA (Schmitt et al. 2002), a model for aIF2 in the ternary initiation complex can be hypothesized (Fig. 5C). Assuming that the acceptor stem of tRNAi is recognized by the -subunit, an interaction of aIF2 with the T-domain is proposed as the size of aIF2 rules out a direct involvement in the recognition of the codon–anticodon interaction. However, there is a clear connection between the recognition of the initiation site (AUG) and the rate of GTP hydrolysis. Affinity labeling with GTP analogs has suggested that eIF2 is in close proximity to the guanine base and ribose moieties of GTP in a region that maps to strands 1 and 2 (Bommer and Kurzchalia 1989; Bommer et al. 1991). At this position, eIF5 presents a well-conserved GNG insertion at the loop connecting these two strands, which may be related to the GAP activities of aIF2, eIF2, and eIF5. It is possible that upon recognition of the initiation codon, some major structural change occurs in the preinitiation complex that trigger the hydrolysis of GTP. This is supported by early work where conformational changes in the tertiary structure of tRNA upon formation of the codon–anticodon interaction were detected (Schwarz et al. 1976; Robertson et al. 1977; Moller et al. 1979). A similar event may occur upon recognition of the initiation codon, where aIF2 could act as an element signal that stimulates GTP hydrolysis.
Initiator tRNAs have several unique sequence and structural characteristics that distinguishes them from elongator tRNAs. For example, the A1:U72 base pair at the end of the acceptor stem and the three consecutive G:C base pairs in the anticodon stem (G29:C41, G30:C40, G31:C39). Initiators also lack the T
C sequence in loop IV of the T-domain containing A54 in place of T54 (of the TC sequence) and A60 instead of pyrimidine-60 (Sprinzl et al. 1998; Hinnebusch 2000). A54, A50:U64, and U51:A63 in the T-region are critical discriminating features, as mutating these residues together with A1:U72 can confer elongator function in vitro (Drabkin and RajBhandary 1998). These sequences are potential targets for aIF2 recognition, and further studies using mutagenesis should elucidate the tRNAi binding properties of aIF2. Most studies on a/eIF2 have focussed on the N-terminal region or the zinc finger; however, further investigations on the HTH motif and the 1–2 turn will provide insight in the role of a/eIF2 in the tRNAi recognition and GTP hydrolysis. The proposed model provides a framework for understanding the processes regulated by a/eIF2 in translation initiation.
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Materials and methods |
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from M. thermoautotrophicum (gene MTH1769) was subcloned into pET15b (Novagen, Inc.) and expressed in E. coli BL21 as an oligo-histidine (His-tag) fusion protein of 161 residues. The molecular weight was confirmed by mass spectrometry. Cells were grown at 37°C to an OD600 of 0.8 and induced with 1 mM IPTG. Afterwards, the temperature was reduced to 30°C and the cells were allowed to express the protein for 3 h before harvesting. The media used were either LB or M9 minimal media containing 15N ammonium chloride and/or 13C-glucose (Cambridge Isotopes Laboratory) and 50 µM ZnCl2. aIF2 was purified by heat denaturation of endogenous E. coli proteins and affinity chromatography on Ni2+-loaded chelating sepharose (Amersham Pharmacia Biotech). The N-terminal His-tag was cleaved from aIF2 by treatment for 24 h at room temperature with thrombin (Amersham Pharmacia Biotech) at 1 unit per mg fusion protein. Benzamidine sepharose was used to remove thrombin. NMR samples were ~1.0 mM protein in 50 mM Bis-Tris buffer, 0.30 M NaCl, 50 µM ZnCl2, 1 mM DTT, 0.02% (w/v) NaN3 at pH 6.0.
NMR spectroscopy
All NMR experiments were recorded at 310 K using standard double and triple resonance techniques on 15N or 13C, 15N-labeled samples (Bax and Grzesiek 1993). All of the experiments were done on Bruker DRX500 and Varian INOVA 800 MHz spectrometers. The following experiments were recorded and evaluated: (1) for backbone assignments: HNCACB and CBCACONH (Grzesiek et al. 1992; Constantine et al. 1993); (2) for side-chain and NOE assignments: from 15N-TOCSY, 15N-edited NOESY, 2D homonuclear NOESY in H2O and D2O; (3) for dihedral angle restraints: 3JHN-H coupling constants were obtained from HNHA experiment (Kuboniwa et al. 1994); (4) for 15N-1H dipolar couplings: an IPAP-HSQC experiment on an isotropic medium and on a sample containing 18 mg/mL Pf1 phage (Hansen et al. 1998; Ottiger et al. 1998); (5) for backbone dynamics: 15N-1H heteronuclear NOE data were measured by taking the ratio of peak intensities from experiments performed with and without 1H presaturation. Hydrogen bond constraints were introduced to secondary structure regions as determined by chemical shift analysis, HNHA experiments, and characteristic NOE patterns. Hydrogen bonds were defined as a restraint from the carbonyl oxygen to the amide hydrogen and nitrogen, using a standard length of 1.8 Å and 2.8 Å, respectively. Additional 
and dihedral restraints were obtained using the TALOS (Cornilescu et al. 1999). All NMR spectra were processed using either XWINNMR version 2.5 or 3.1 (Bruker Biospin) or GIFA (Malliavin et al. 1998). Evaluation of spectra and manual assignments were completed with XEASY (Bartels et al. 1995).
Analysis and structure calculations
CNS 1.1 software (Brunger et al. 1998) was used to generate an initial fold of aIF2 with a basic set of NOEs acquired from manual assignments of 3D 15N-edited NOESY and 2D homonuclear NOE spectra including dihedral angle and hydrogen bond constraints (Wüthrich 1986). These calculations generated a fold that was used as a model template for automated assignments by ARIA1.1 (Nilges et al. 1997). The final structure of aIF2 was calculated with a total set of 835 constraints (Table 1) collected from the experiments described earlier. In the final round of calculations, CNS 1.1 was extended to incorporate RDC restraints for further refinement, using the torsion angle space. The axial and rhombic components of the alignment tensor were defined from a histogram of measured RDCs (Clore et al. 1998a) and optimized by a grid search method (Clore et al. 1998b). Refinement of the whole protein using a single alignment tensor resulted in poor fits, which may reflect interdomain motion. We therefore proceeded to refine the structure using two separate alignment tensors to define each well-structured domain. The 20 structures were selected based on the lowest overall energy and least violations to represent final structures. PROCHECK was used to generate Ramachandran plots to check the protein’s stereochemical geometry (Laskowski et al. 1993). The coordinates of aIF2 have been deposited in the RCSB under PDB code 1NEE and the NMR assignments under BMRB accession 4385
[BMRB]
.
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Acknowledgments |
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and D. Elias, G. Kozlov, T. Sprules, A. Denisov, N. Lim, C. Gauthier, M. Bachetti, C. Deprez, and I. D’Orso for assistance and helpful discussions. The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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