A Conserved HEAT Domain within eIF4G Directs Assembly of the Translation Initiation Machinery

Molecular Cell, Vol. 7, 193–203, January, 2001, Copyright 2001 by Cell Press

A Conserved HEAT Domain within eIF4G Directs Assembly of the Translation Initiation Machinery Joseph Marcotrigiano,* Ivan B. Lomakin,‡ Nahum Sonenberg,§ Tatyana V. Pestova,‡ Christopher U. T. Hellen,‡ and Stephen K. Burley*†k * Laboratories of Molecular Biophysics † Howard Hughes Medical Institute The Rockefeller University New York, New York 10021 ‡ Department of Microbiology and Immunology Morse Institute for Molecular Genetics State University of New York Health Science Center at Brooklyn Brooklyn, New York 11203-2098 § Department of Biochemistry and McGill Cancer Center McGill University Montreal, Quebec Canada H3G 1Y6

Summary The X-ray structure of the phylogenetically conserved middle portion of human eukaryotic initiation factor (eIF) 4GII has been determined at 2.4 A˚ resolution, revealing a crescent-shaped domain consisting of ten ␣ helices arranged as five HEAT repeats. Together with the ATP-dependent RNA helicase eIF4A, this HEAT domain suffices for 48S ribosomal complex formation with a picornaviral RNA internal ribosome entry site (IRES). Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of this essential component of the translation initiation machinery that, respectively, bind eIF4A and a picornaviral IRES. The structural and biochemical results provide mechanistic insights into both capdependent and cap-independent translation initiation. Introduction eIF4G (eukaryotic initiation factor 4G) is a modular adaptor protein that plays a pivotal role in coordinating the assembly of translation factors and the small ribosomal subunit during the rate-limiting initiation stage of protein synthesis (Figure 1; for comprehensive reviews, see Hentze, 1997; Morley et al., 1997; Gingras et al., 1999a). This central component of the translation machinery contains a poly(A) binding protein (PABP) interacting motif (Tarun and Sachs, 1996; Imataka et al., 1998), a Tyr-X4-Leu-⌽ (⌽ denotes a hydrophobic amino acid) eIF4E-recognition motif (Mader et al., 1995), and a phylogenetically conserved middle segment responsible for binding eIF4A and type II picornaviral RNA IRESs (internal ribosome entry sites; Lamphear et al., 1995; Pestova et al., 1996b; Imataka and Sonenberg, 1997) (Figure 1). Mammals possess two functional isoforms of eIF4G k To whom correspondence should be addressed (e-mail: burley@ rockvax.rockefeller.edu).

(eIF4GI and eIF4GII), with an additional C-terminal region that contains a second eIF4A binding site and a segment that interacts with a physiologic eIF4E kinase, Mnk-1 (Figure 1) (Lamphear et al., 1995; Imataka and Sonenberg, 1997; Pyronnet et al., 1999). Ribosome binding experiments with ␤-globin mRNA have demonstrated that the eIF4E-recognition motif and the middle portion of eIF4G constitute a minimal eIF4G core, which is capable of supporting cap-dependent translation initiation in vitro (Morino et al., 2000). It is also remarkable that a chimeric protein consisting of the RNA binding region of iron response protein (IRP-1) fused to the eIF4A/IRES binding domain of eIF4G can direct translation of a cistron bearing an upstream iron-responsive element in a cap-independent manner (De Gregorio et al., 1999). Given its critical role in coordinating the assembly of the translation machinery, it is not surprising that eIF4G constitutes an important target for regulation of protein synthesis. The mammalian eIF4E binding proteins (4EBPs) and yeast p20 inhibit cap-dependent translation by binding to eIF4E and preventing formation of the eIF4E–eIF4G–eIF4A heterotrimer, known as eIF4F (Gingras et al., 1999a). Biochemical and X-ray crystallographic studies have documented that the 4E-BPs also possess Tyr-X4-Leu-⌽ eIF4E-recognition motifs (Figure 1), which compete with the same motif in eIF4G for binding to the convex dorsal surface of eIF4E (Haghighat et al., 1995; Marcotrigiano et al., 1999). The inhibitory effect of the 4E-BPs is relieved by phosphorylation in response to growth stimulatory signals received at the cell surface (Pause et al., 1994; Gingras et al., 1999b). Mammalian eIF4Gs have two distant orthologs, p97 (Imataka et al., 1997; also known as NAT1 [Yamanaka et al., 1997], DAP-5 [Levy-Strumpf et al., 1997], and eIF4G2 [Shaughnessy et al., 1997]) and a poly(A) binding protein interacting protein (Paip-1) (Craig et al., 1998). p97 resembles the C-terminal two thirds of human eIF4G (Figure 1) and binds to eIF4A and the 43S ribosomal complex (consisting of the 40S ribosomal subunit, Met-tRNAiMet, eIF2, eIF3, and eIF1), but not to eIF4E or PABP. In vitro and in vivo experiments have shown that p97 inhibits protein synthesis (Imataka et al., 1997), which may be due to sequestration of the 43S ribosomal complex and/ or eIF4A. Paip-1, the more distant of the two eIF4G relatives, resembles the phylogenetically conserved middle region of eIF4G and contains a C-terminal PABP binding site (Figure 1). Overexpression of Paip-1 in tissue culture increases the rate of translation initiation, suggesting an alternative mechanism to circularize mRNA and increase translation efficiency (Craig et al., 1998). Modulation of eIF4G activity also has pronounced effects on translation initiation and cellular behavior. Simultaneous disruption of both eIF4G genes in yeast is lethal (Goyer et al., 1993). Overexpression of eIF4G in NIH-3T3 cells results in a malignant phenotype (FukuchiShimogori et al., 1997), and abnormally high levels of eIF4G have been found in some melanomas (Brass et al., 1997). Exposure of human B and T lymphoid tumor cells to conditions that inhibit cell proliferation and in-

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Figure 1. Motifs Common to eIF4GI, eIF4GII, and eIF4G-Related Proteins (Left) Schematic alignment of the amino acid sequences of various eIF4Gs and eIF4G-related proteins, highlighting conserved protein binding motifs. Sequence identities of the conserved eIF4A/IRES binding domains with eIF4GII are given as percentages. Binding sites for various proteins are color coded as follows: (PABP), blue; (eIF4E), orange; (eIF4A) central, red; (eIF4A) C-terminal, magenta; (Mnk-1), black. Blue arrow denotes picornaviral protease cleavage site. (Right) Schematic diagram contrasting cap-dependent translation initiation from cap-independent, IRES-mediated translation initiation from a picornaviral IRES.

duce apoptosis leads to degradation of eIF4G (Clemens et al., 1998). Most picornaviral infections (all genera except cardiovirus and hepatovirus) cause specific proteolytic cleavage of the eIF4Gs, separating the Tyr-X4Leu-⌽ eIF4E-recognition motif and PABP binding site from the eIF4A/IRES binding sites (blue arrows, Figure 1). In contrast, cardioviruses (including encephalomyocarditis virus, EMCV) inhibit cellular translation by dephosphorylating 4E-BP1, thereby disrupting the eIF4E/ eIF4G complex required for assembly of eIF4F (Gingras et al., 1996). Silencing of cap-dependent translation during picornaviral infections permits exploitation of the host cell translation machinery for synthesis of viral proteins. Picornaviral genomes are linear, positive-sense, singlestranded RNAs with a 3⬘ poly(A) tail and a viral protein (VPg) covalently attached to the 5⬘ end (instead of a 7-methyl-G cap). These viral genome 5⬘ UTRs contain highly structured RNA segments or IRESs that recruit the ribosome directly to the initiation codon (Jang et al., 1988; Pelletier and Sonenberg, 1988). Picornaviral IRESs can be grouped into two different categories (type I enterovirus and rhinovirus, and type II cardiovirus and aphthovirus) based on primary sequence and predicted secondary structure (Jackson and Kaminski, 1995). Reconstitution assays have shown that cap-independent translation from the EMCV (type II) IRES utilizes a subset of the canonical translation initiation factors required for cap-dependent protein synthesis (Pestova et al., 1996a). Further biochemical and mechanistic studies of type II IRESs have demonstrated that the middle region of hu-

man eIF4GI (residues 613–1090) recognizes a structured element within the IRES, located immediately upstream of the translation start site (Ohlmann et al., 1996; Pestova et al., 1996b; Kolupaeva et al., 1998; Pilipenko et al., 2000). Together with eIF4A, eIF4G and the IRES recruit the 43S ribosomal complex to the viral RNA (Lomakin et al., 2000), yielding a 48S ribosomal complex. Subsequent binding of the 60S ribosomal subunit permits translation of the viral polyprotein. Here, we present the X-ray crystal structure of the phylogenetically conserved middle domain of human eIF4GII (745–1003), which is responsible for interactions with eIF4A and type II picornaviral IRESs. The crescentshaped protein is composed of ten ␣ helices arranged as five antiparallel ␣ helical pairs or HEAT repeats. Functional studies were used to identify surface features responsible for interactions with eIF4A and the EMCV IRES, providing insights into the mechanisms of both cellular (cap-dependent) and viral (cap-independent) translation initiation. Results and Discussion Crystallization and Structure Determination Sequence comparisons (Figure 2A), secondary structure predictions, and proteolysis combined with mass spectrometry (data not shown) permitted identification of a protease-resistant portion of human eIF4GII (745–1003) that supports binding to eIF4A and the EMCV IRES (Figure 2B). Purified preparations of eIF4GII (745–1003) yielded high-quality crystals, which contain one pro-

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Figure 2. Structural and Functional Characterization of the eIF4A/IRES Binding Domain of Human eIF4G (A) Structure-based sequence alignments of the eIF4A/IRES binding domains of all known eIF4Gs and eIF4G-related proteins, including human eIF4GII (Gradi et al., 1998) (used in structure determination), human eIF4GI (Imataka et al., 1998), Saccharomyces cerevisiae TIF4631 and TIF4632 (Goyer et al., 1993), Triticum aestivum eIF-(iso)4G (Allen et al., 1992), human p97 (Imataka et al., 1997), and human Paip-1 (Craig et al., 1998). ␣-helical secondary structural elements are denoted with cylinders and labeled with repeat number and a or b. Dashed lines correspond to regions with poorly resolved electron density. Color scheme: red, identity; blue, high conservation. V8 protease cleavage sites are denoted by arrows. Location of eIF4G mutants: (1), M-1 (Imataka and Sonenberg, 1997); (4), M-4 (Imataka and Sonenberg, 1997); (7), mut(Ile749→Thr, Arg754→Ile) (Lomakin et al., 2000); (8), mut796-Ins8 (Lomakin et al., 2000); (R), RRM1 (Lomakin et al., 2000); (t), tif4632–6 (Neff and Sachs, 1999); (A), reduced eIF4A binding, 756; (K), reduced IRES binding, 834; (B), reduced eIF4A and IRES binding, 814; (N), no effect, 798, 802, 803, 843, and 888. (See Table 2 for a complete description of eIF4G mutations.) (B) Toeprint analyses of the interaction of eIF4GII (extended construct: 735–1097 and crystallization construct: 745–1003) with the EMCV IRES in the presence and absence of eIF4A. The full-length cDNA extension product is marked (e). A common stop site within the EMCV RNA used as an internal normalization standard is denoted by (N). The stop site due to binding of eIF4GII is indicated by (C786). Reference lanes (t), (c), (g), and (a) depict the EMCV IRES cDNA sequence.

tomer per asymmetric unit (see Experimental Procedures). The structure was determined via multiwavelength anomalous dispersion (MAD) (Hendrickson, 1991) (Table 1). Experimental phases obtained at 2.4 A˚ resolution yielded a high-quality electron density map, which was further improved by density modification and phase combination. The current refinement model has an R factor of 23.9% and a free R value of 28.5% (Bru¨nger, 1992) at 2.37 A˚ resolution with excellent stereochemistry (Table 1). Structural Overview The three-dimensional structure of the middle domain of human eIF4GII (745–1003) is illustrated schematically in Figure 3. The crescent-shaped molecule consists of ten ␣ helices with overall dimensions 50 A˚ (length) ⫻ 20 A˚ (depth) ⫻ 40 A˚ (width). The polypeptide chain forms a right-handed solenoid, with its superhelical axis perpendicular to the cylindrical axes of the ␣ helices. The

crescent is generated by five repeating pairs of antiparallel ␣ helices, stacked one repeat upon the other. The ten ␣ helices are arranged in the order 1a-1b-2a-2b3a-3b-4a-4b-5a-5b (numbers denote each pair and the underlined, lowercase letters refer to individual ␣ helices within each pair). This repeating pattern gives rise to a double layer of ␣ helices with the convex and concave surfaces formed by the a and b ␣ helices, respectively. The intrarepeat and interrepeat loops segregate to opposite surfaces of the crescent-shaped molecule (Figure 3B). For simplicity, we will refer to the molecular surfaces of the eIF4GII crescent as concave (b ␣ helices, right in Figure 3A), convex (a ␣ helices, left in Figure 3A), interrepeat (left in Figure 3B), and intrarepeat (right in Figure 3B). Adjacent repeats are stabilized by salt bridges and Van der Waals interactions, which run the length of the polypeptide chain, giving rise to an extended hydrophobic core. ␣ helices within repeats 1–2 and 3–5 are ar-

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Table 1. Statistics of the Crystallographic Analysis Experimental MAD Statistics (7 Se Sites) Data Set

Resolution (A˚)

Reflections Measured/Unique

Completeness (%) Overall/Outer Shell

Rsym (%) Overall/Outer Shell

␭1 (0.97928 A˚) ␭2 (0.97908 A˚) ␭3 (0.93922 A˚) Overall Figure of Merit Acentric Centric

20.0–2.37 20.0–2.37 20.0–2.37

170,724/12,425 170,252/12,394 169,370/12,401

97.5/85.0 96.6/80.0 97.7/87.4

4.4/19.5 4.7/21.3 4.3/20.8

Resolution (A˚)

Completeness (%)

R Factor

Free R Factor

20.0–2.37

97.5

0.239

0.285

Phasing Power Ano

Iso

3.21 4.01 3.16

— 1.63 2.18

0.64 0.00

Refinement Statistics (against ␭1) Data with 兩F兩 ⬎ 2␴(兩F兩) Rms Deviations Bond lengths Bond angles Thermal parameters

0.008 A˚ 1.7⬚ 1.0 A˚2

Rsym ⫽ 兺|I⫺⬍I⬎|/兺I, where I ⫽ observed intensity, ⬍I⬎ ⫽ average intensity obtained from multiple observations of symmetry-related reflections. Phasing power ⫽ rms (兩FH|/E), where 兩FH| ⫽ heavy atom structure factor amplitude, and E ⫽ residual lack of closure. Rms bond lengths and rms bond angles are the respective root-mean-square deviations from ideal values. Rms thermal parameter is the root-mean-square deviation between the B values of covalently bonded atomic pairs. Free R factor was calculated with 10% of data omitted from the structure refinement.

ranged in a parallel stack with a rotation of less than 25⬚ between neighboring repeats about the solenoid axis. The crescent shape arises from a right-handed 50⬚ rotation of repeat 3 relative to repeat 2. The 2b-3a and 3a-3b loops located at the site of this large rotation do not have well-defined electron density. There is no evidence that the relative orientations of these two subregions are likely to change upon higher-order complex formation (buried contact surface area between each pair of successive a-b ␣-helical repeats ⫽ 510–730 A˚2). Figure 2A documents that residues conserved among the middle portions of all eIF4Gs and their orthologs map to the ␣ helices observed in the structure of eIF4GII (745–1003). The sites of insertions and deletions within individual sequences correspond to random coil portions of our structure, where they should not disrupt ␣ helix formation. The level of amino acid sequence identity (23%–88%) and the pattern of amino acid differences across phyla allow us to conclude that this region of all eIF4Gs (including yeast) and the eIF4G-related proteins (p97 and Paip-1) share the same three-dimensional structure (Sander and Schneider, 1991). Structural Comparisons of the Middle Domain of eIF4GII with Two Canonical HEAT Repeat Proteins DALI server (Holm and Sander, 1993) comparisons of the structure of the middle domain of eIF4GII with the contents of the Protein Data Bank (PDB, http://www. rcsb.org/) revealed similarities to various HEAT repeat proteins (named for Huntingtin, Elongation factor 3, A subunit of protein phosphatase 2A [PP2A], and Target of rapamycin). PR65/A subunit of PP2A (Groves et al., 1999; PDB code 1b3u; Z score ⫽ 9.1 and root-meansquare deviation (rmsd) ⫽ 4.7 A˚) and the nuclear import factor, importin ␤, (Cingolani et al., 1999; Chook and Blobel, 1999; PDB codes 1qgr and 1qbk; Z scores ⫽ 9.0 and 8.1 and rmsds ⫽ 5.8 A˚ and 2.9 A˚, respectively) represent the closest structural homologs of the middle domain of eIF4GII. HEAT repeat proteins participate in a wide variety of cellular processes that are dependent

on assembling large multiprotein complexes (Andrade and Bork, 1995). The HEAT repeat itself consists of two antiparallel ␣ helices of varying length (designated a and b) that occur in tandem arrays repeated 3–22 times, with an average of 14 repeats per member (Andrade and Bork, 1995). As a general rule, HEAT repeat proteins do not share any absolutely conserved amino acids. Family members were initially identified using secondary structure predictions, which yielded similar patterns of hydrophobic residues with a highly conserved Pro within each a ␣ helix (Andrade and Bork, 1995). Hydrophobic residues line one face of each of the ␣ helices to form an extended hydrophobic core, which is created by approximation of successive a plus b ␣-helical pairs. The conserved Pro causes a disruption of the hydrogen bonding network within ␣ helix a, producing a marked bend (typically about 45⬚). Many of the b ␣ helices are also bent, although this feature is not correlated with the presence of a conserved Pro. Figure 4 demonstrates that HEAT repeats 2–6 of PR65/A and 10–14 of importin ␤ also form molecular crescents (left, convex surface; right, concave surface). The arrangement of ␣ helices in eIF4GII repeats 2–5 resembles the arrangements seen in repeats 3–6 of PR65/A and 11–14 of importin ␤. The most significant difference between the middle domain of eIF4GII and these two canonical HEAT repeat proteins involves the linearity of the ␣ helices. A majority of ␣ helices within PR65/A and importin ␤ are bent, whereas the eIF4GII ␣ helices lack Pro residues and are predominantly straight. Despite these minor structural differences, we believe that eIF4GII, eIF4GI, p97, and Paip-1 represent previously unidentified members of a HEAT repeat protein superfamily that contribute to the assembly of the protein synthesis machinery. Identification of eIF4A- and EMCV IRES–Binding Sites on the Molecular Surface of eIF4GII Our structure of the eIF4A/IRES binding domain of eIF4GII provides a rational basis for directed studies of

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Figure 3. Structure of the eIF4A/IRES Binding Domain of eIF4GII (A) Ribbon stereodrawing of the conserved central region of eIF4GII viewed along the cylindrical axes of the ␣ helices, with the concave surface on the right and the convex surface on the left. ␣ helices are labeled as in Figure 2A. (B) Stereodrawing viewed perpendicular to the ␣ helix axes, rotated 90⬚ about the solenoid axis relative to (A), with the concave surface in the foreground. The intra- and interrepeat surfaces are located on the right and left, respectively. (C) Stereodrawing viewed along the ␣-helix axes, rotated 180⬚ about the solenoid axis relative to A, with the intrarepeat surface in the foreground and the concave and convex surfaces on the left and right, respectively.

higher-order complex formation during cap-dependent and cap-independent translation. Mapping of phylogenetically conserved, solvent-accessible residues proved a useful method of identifying potential translation factor binding sites on the surfaces of both eIF4E and PABP (Marcotrigiano et al., 1997, 1999; Deo et al., 1999). The middle domain of eIF4G does not, however, display any convincing clusters of conserved surface features (data not shown). In an attempt to characterize the eIF4A- and IRES binding sites, we performed site-directed mutagenesis of various solvent-accessible residues to change the chemical properties of the protein surface without dis-

rupting the fold of the crescent-shaped domain (enumerated in Table 2). Previous biochemical studies have demonstrated that both eIF4GI (613–1090) and eIF4A are required for high-affinity binding to the EMCV IRES, thereby promoting formation of a 48S ribosomal complex on the EMCV RNA (Pestova et al., 1996b; Lomakin et al., 2000). Binding of various surface mutants of eIF4GII to the EMCV IRES (both with and without eIF4A) was detected by inhibition of primer extension (Figures 5A and 5B) (Pestova et al., 1996a). Mutants 814 and 834 exhibit a dramatic reduction in IRES binding, which was unaffected by addition of eIF4A. Mutants 756 and 798 display a modest decrease in IRES binding activity (Fig-

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Figure 4. Comparison of the Middle Domain of eIF4GII with Other HEAT Repeat Proteins Tubular ␣ helix drawing comparing eIF4GII (745–1003) with the PR65/A subunit of PP2A (40–235) (Groves et al., 1999) and importin ␤ (401–640) (Cingolani et al., 1999), using the view in Figure 3A (concave surface, right). Protein superpositions were determined by the DALI server (Holm and Sander, 1993). For ease of comparison, ␣ helices are numbered as in Figure 2A.

ure 5C). Addition of eIF4A to the 798 mutant restores wild-type IRES binding activity, whereas the phenotype of mutant 756 is unaffected by eIF4A (Figures 5A–5C). In addition, the eIF4GII mutants were assayed for binding to eIF4A (Figure 5D). M-1, a previously characterized eIF4GI mutant with impaired eIF4A binding activity, was used as a negative control (Imataka and Sonenberg, 1997). Mutants 756 and 814 show dramatically reduced eIF4A binding, while mutants 834, 798, and 803 have little or no effect. In summary, we have identified two surface mutants (756 and 834) with decreased affinity for eIF4A and EMCV IRES, respectively, and one mutant (814) deficient in binding to both eIF4A and the EMCV IRES. The locations of mutations that affect eIF4A and/or EMCV IRES binding are illustrated in Figures 2A and 6. In addition, we have included findings from previously published site-directed mutagenesis studies of human and yeast eIF4Gs (Imataka and Sonenberg, 1997; Neff and Sachs, 1999; Lomakin et al., 2000; Morino et al., 2000) and human p97 (Imataka and Sonenberg, 1997) that were performed in advance of our structure determination. Knowledge of the three-dimensional structure allowed us to focus on surface changes that affect the eIF4A and/or EMCV IRES–binding properties of eIF4G. (Deleterious mutations within the hydrophobic core were not considered because they almost certainly destabilize protein structure.) Taken together, the eIF4G mutagenesis results permitted identification of two distinct molecular surfaces that respectively support interactions with eIF4A and the EMCV IRES. The entire length of the intrarepeat face of the crescent (Figures 3C and

6C, black) is responsible for recognition of eIF4A. The EMCV IRES–binding surface maps to the interrepeat face overlying repeats 1, 2, and 3 (Figures 3A and 6A, cyan). Regrettably, our X-ray structure did not provide any information regarding the positions of residues 823– 831, which were not visible in the electron density maps and are presumed disordered in our crystals. Insertion of an octapeptide sequence (Glu-Gly-Glu-Gln-Gly-GluAla-Gly) into the corresponding region of human eIF4GI (between Val-796 and Thr-797) demonstrated that the surface loop connecting ␣ helices 2b and 3a is important for interactions with both eIF4A and the EMCV IRES (Lomakin et al., 2000). Figures 6A (right panel) and 6C (right panel) show the location of this disordered loop (denoted by closed dots), identifying one intersection/ overlap area of the eIF4A- and EMCV IRES–binding surfaces. A double point mutant (Arg-814→Asp, Lys820→Asp) has the same phenotype, revealing a second region where the two interaction surfaces intersect/ overlap. Figure 6 (left panels) illustrates the solvent-accessible surface of the middle domain of eIF4GII color-coded for electrostatic potential. The IRES binding surface (Figure 6A, right panel, cyan) corresponds to a positively charged/hydrophobic surface that may be responsible for phosphate neutralization and base recognition during IRES binding (Figure 6A, left panel). The eIF4A binding site is highly polar, suggesting that the protein– protein interaction involves hydrogen bonds and/or salt bridges. Earlier biochemical studies of eIF4F demonstrated disruption of the eIF4A-eIF4G interaction during anion exchange chromatography (Etchison and Milburn,

Table 2. Surface Mutations in the Middle Domain of eIF4GII Mutant Identifier

Amino Acid Substitution in eIF4GII (737–1097)

756 798 802 803 814 834 843 888

Arg-756→Asp, Arg-759→Asp, Lys-764→Asp Lys-798→Ala Glu-802→Ala Pro-803→Ala Arg-814→Asp, Lys-820→Asp Arg-834→Asp, Lys-835→Asp Lys-843→Asp Arg-888→Ala

(A) (N) (N) (N) (B) (K) (N) (N)

Letters within parentheses following each mutant identifier denote the effect of the mutation: (A), reduced eIF4A binding; (K), reduced IRES binding; (B), reduced eIF4A and IRES binding; (N), no effect as in Figure 2A.

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Figure 5. Site-Directed Mutagenesis of eIF4GII (A and B) Toeprint analyses of the interaction of eIF4GII (735–1097) mutant polypeptides with the EMCV IRES alone (A) and with added eIF4A (B). (C) Histogram showing the results of quantitative toeprinting of the EMCV RNA with eIF4GII. Solid (eIF4G) and open (eIF4G and eIF4A) bars represent average values obtained in five replicate experiments. (D) Interaction of mutant eIF4GII (735–1097) polypeptides with immobilized eIF4A in a direct binding assay. eIF4A was visualized by Coomassie staining, and eIF4GII polypeptides were detected by Western blotting with anti-T7 tag antibodies. (E) Primer extension analyses of 48S initiation complexes assembled on EMCV RNA using purified eIF1, eIF1A, eIF2, eIF3, eIF4A, eIF4B, methionyl initiator tRNA, and 40S ribosomal subunits with wild-type or mutant eIF4GII. The full-length cDNA extension product is marked (e), the position of the stop site due to binding of eIF4GII is denoted by (C786), and cDNA products labeled (AUG826) and (AUG834) terminated at stop sites 15–17 nt downstream of each initiation codon. Reference lanes (t), (c), (g), and (a) depict the EMCV IRES cDNA sequence.

1987), which is consistent with largely polar protein– protein interactions. We also assayed all of our eIF4GII mutants for 48S ribosomal complex formation with EMCV RNA. Mutants defective in binding to eIF4A and/or the EMCV IRES (756, 814, and 834) do not support 48S complex forma-

tion (Figure 5E). All of our structure-based eIF4GII mutations were tested in the context of a C-terminal extension (residues 735–1097) beyond the confines of our crystallization construct of eIF4GII to ensure efficient interaction with eIF3 (Experimental Procedures). We subsequently showed, however, that the shorter eIF4GII

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Figure 6. Surface Properties of eIF4A/IRES Binding Domain of eIF4GII GRASP (Gilson et al., 1988) representation of the solvent-accessible surface of the eIF4GII calculated using a water probe radius of 1.4 A˚. (Left) The surface color-coded for calculated electrostatic potential: red ⬍ ⫺9kBT and blue ⬎ 9kBT, where kB denotes Boltzmann constant and T, temperature. (Right) The surface color-coded for the locations of mutants affecting binding to eIF4A (black) and EMCV IRES (cyan). The disordered 2b-3a loop (residues 823–831) is represented by closed dots. (A), (B), and (C) views are identical to those shown in Figures 3A, 3B, and 3C, respectively.

fragment in our crystals supports EMCV IRES binding (Figure 2B) and 48S ribosomal complex formation with the same IRES (Figure 5E). A similar finding was recently reported for the corresponding region of eIF4GI (697– 970) (Lomakin et al., 2000). It is possible that a surface region of eIF4G in the vicinity of conserved Glu-802 and Pro-803 (eIF4GII numbering) is responsible for ribosome recruitment, because mutants 802 and 803 show modest defects in 48S ribosomal complex formation, without affecting binding to eIF4A or the EMCV IRES (Figure 5E).

We conclude, therefore, that mechanistic studies based on our structure of the middle domain of eIF4GII should provide significant insights into the process of 43S ribosomal complex recruitment by both cellular and viral mRNAs. Conclusion We have determined the X-ray crystal structure of an unexpected HEAT repeat domain for the phylogenetically conserved middle portion of eIF4G, which serves

eIF4G Binds eIF4A/IRES via HEAT Domain 201

as a central assembly platform at the heart of the eukaryotic translation initiation machinery. Site-directed mutagenesis and biochemical studies have allowed us to locate conserved, adjacent binding surfaces for eIF4A and a picornaviral type II IRES, explaining synergistic interactions among eIF4G, eIF4A, and the IRES. Our work provides a starting point for further systematic biochemical, genetic, and structural studies aimed at understanding interactions of eIF4G with eIF4A, picornaviral type II IRESs, mRNA, and other components of the 43S ribosomal complex. Experimental Procedures Protein Preparation and Crystallization Human eIF4GII (residues 745–1003) was expressed in E. coli with an N-terminal, hexahistidine tag and purified via Ni2⫹ affinity chromatography using standard methods, followed by overnight thrombin cleavage at 4⬚C. Purification to homogeneity involved subtractive Q Sepharose chromatography, followed by heparin affinity chromatography. The resulting protein was neither proteolyzed nor modified as confirmed by matrix-assisted laser desorption ionization mass spectrometry (measured mass ⫽ 30,864 ⫾ 40; predicted mass ⫽ 30,834). Dynamic light scattering by a DynaPro Molecular Size Detector (Protein Solutions Inc., Charlottesville, VA) demonstrated that the protein is monomeric and monodisperse at 1 mg/mL. Proteolysis by chymotrypsin and trypsin failed to produce any fragments, whereas prolonged exposure to V8 produced fragments that were analyzed by N-terminal sequencing and mass spectrometry. eIF4GII (745–1003) crystals were obtained by hanging drop vapor diffusion against a reservoir of 0.1 M acetate (pH 4.6), 0.1 M ammonium acetate, 20%–22% (w/v) PEG 4000, and 5 mM tris(carboxyethyl)phospine at 4⬚C. The crystals belong to the trigonal space group R3 (unit cell: a ⫽ 118.8 A˚, c ⫽ 58.2 A˚, hexagonal axes) with one molecule per asymmetric unit, and they diffract to Bragg spacings of better than 2.0 A˚. eIF4GII (745–1003) was expressed in its selenomethionine-substituted form and purified, characterized, and crystallized as above. Crystal cryoprotection prior to quick freezing by immersion in liquid propane was achieved by slow transfer to 0.1 M acetate (pH 4.6), 0.1 M ammonium acetate, 22% (w/v) PEG 4000, and 15% (v/v) glycerol. Data Collection and Structure Determination Se-Met multiwavelength anomalous dispersion data (Hendrickson, 1991) were collected from a single frozen crystal at Beamline X25 (National Synchrotron Light Source, Brookhaven National Laboratory) at three X-ray wavelengths in the vicinity of the Se K absorption edge (Table 1). Data were processed and scaled using DENZO/ SCALEPACK (Otwinowski and Minor, 1997). Seven of eight possible selenium sites were found by SOLVE (Terwilliger and Berendzen, 1997), and an interpretable electron density map was obtained using SHARP (de La Fortelle and Bricogne, 1997) followed by density modification and phase combination by DM (Collaborative Computational Project Number 4, 1994). Several rounds of iterative model building and refinement were performed using O (Jones et al., 1991) and CNS (Bru¨nger et al., 1998). The current refinement model contains residues 745–822, 832–850, 881–986, 65 water molecules, and one additional amino acid on the N terminus derived from the expression vector, giving a final R factor of 23.9% and free R factor of 28.5% with excellent stereochemistry (Table 1). The mean thermal factors are 21 A˚2 for the protein and 25 A˚2 for solvent molecules, with a rmsd of 1.0 A˚2 between the B factors of covalently bonded atoms. PROCHECK (Laskowski et al., 1993) revealed no unfavorable (φ,␺) combinations, with main chain and side chain structural parameters consistently better than average (overall G value ⫽ 0.3). Construction and Assay of eIF4G Mutants for eIF4A and EMCV IRES Binding pFLAG(HIS)6-eIF4A was constructed by cloning cDNA corresponding to the complete eIF4A coding sequence product into pFLAGMAC. peIF4GI(697–1076) (M-1) contains a triple substitution (Leu729→Ala, Leu-732→Ala, Pro-737→Ala) and was made by inserting cDNA corresponding to amino acids 697–1076 derived by PCR from

the eIF4G1 M-1 mutant (Imataka and Sonenberg, 1997). All eIF4GII mutants were obtained by PCR mutagenesis. PCR products were cloned in BamHI–XhoI restriction sites of pET28b and confirmed by sequencing. All proteins were expressed in E. coli and purified via Ni2⫹ affinity chromatography using standard methods. Aliquots (4 ␮g) of FLAG (His)6-eIF4A were immobilized on anti-FLAG agarose beads by incubation at 26⬚C for 20 min with mixing. Beads were washed twice with cold buffer (150 mM NaCl, 10 mM Na2HPO4, 4 mM NaH2PO2 [pH 7.3], 0.1% Triton X-100) to remove unbound protein, mixed with ⵑ3 ␮g of wild-type or mutant eIF4GII (735–1097) or eIF4GI (697–1076) (M-1) proteins with 20 ␮g of BSA and 40 ␮g of RNaseA, incubated at 26⬚C for 20 min with occasional mixing, and washed four times with cold buffer. Proteins were resolved by SDS–PAGE and detected by either Coomassie staining (eIF4A) or Western analysis (eIF4GI and eIF4GII) using an anti-T7-tag HRP conjugate. The specific interaction of recombinant wild-type and mutant forms of eIF4GII (735–1097) with the EMCV IRES was assayed by primer extension inhibition (toe-printing), as described previously (Pestova et al., 1996a). Acknowledgments We thank Drs. L. Berman, J.B. Bonanno, H.A. Lewis, and K. Musunuru for invaluable assistance with X-ray measurements, and Drs. R.C. Deo, A.-C. Gingras, D. Jeruzalmi, V. Kolupaeva, J. Kuriyan, G.A. Petsko, and M. Romanowski for many useful discussions. We thank Ms. T.B. Niven for editorial assistance. S. K. B. is an Investigator for the Howard Hughes Medical Institute, and N.S. is a Howard Hughes Medical Institute International Scholar. This work was supported by Human Frontiers Scientific Program grant RG0303 (S. K. B., N. S., and Edward Darzynkiewicz); grant GM61262 from the National Institutes of Health (S. K. B.); and grant MCB-9726958 from the National Science Foundation (C. U. T. H.). J. M. was supported by a Burroughs-Wellcome Fund Interfaces Program graduate fellowship and a David Rockefeller graduate fellowship. Received September 19, 2000; revised November 28, 2000. References Allen, M.L., Metz, A.M., Timmer, R.T., Rhoads, R.E., and Browning, K.S. (1992). Isolation and sequence of the cDNAs encoding the subunits of the isozyme form of wheat protein synthesis factor 4F. J. Biol. Chem. 267, 23232–23236. Andrade, M.A., and Bork, P. (1995). HEAT repeats in the Huntington’s disease protein. Nat. Genet. 11, 115–116. Brass, N., Heckel, D., Sahin, U., Pfreundschuh, M., Sybrecht, G.W., and Meese, E. (1997). Translation initiation factor eIF-4gamma is encoded by an amplified gene and induces an immune response in squamous cell lung carcinoma. Hum. Mol. Genet. 6, 33–39. Bru¨nger, A.T. (1992). Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature 355, 472–475. Bru¨nger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography and NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. 54, 905–921. Chook, Y.M., and Blobel, G. (1999). Structure of the nuclear transport complex karyopherin-beta2-Ran x GppNHp. Nature 399, 230–237. Cingolani, G., Petosa, C., Weis, K., and Muller, C.W. (1999). Structure of importin-beta bound to the IBB domain of importin-alpha. Nature 399, 221–229. Clemens, M.J., Bushell, M., and Morley, S.J. (1998). Degradation of eukaryotic polypeptide chain initiation factor (eIF) 4G in response to induction of apoptosis in human lymphoma cell lines. Oncogene 17, 2921–2931. Collaborative Computational Project Number 4. (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D50, 760–763. Craig, A.W., Haghighat, A., Yu, A.T., and Sonenberg, N. (1998). Interaction of polyadenylate-binding protein with the eIF4G homologue PAIP enhances translation. Nature 392, 520–523.

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