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ARTICLE Investigation of the conserved glutamate immediately following the DEAD box in eukaryotic translation initiation factor 4AI Krishnaben Patel, Grishma K. Shah, Sai Shilpa Kommaraju, and Woon-Kai Low

Abstract: The DExD-box family (DEAD-box) of proteins was surveyed for eukaryotic translation initiation factor 4A-specific sequences surrounding the DEAD box. An eIF4A-unique glutamate residue (E186 in eIF4AI) was identified immediately following the D-E-A-D sequence in eIF4AI, II, and III that was found to be conserved from yeast to Man. Mutation to a selection of alternative amino acids was performed within recombinant eIF4AI expressed in Escherichia coli and mutant proteins were surveyed for RNA-dependent ATPase activity. The mutants were also investigated for changes in activity in the presence of the two eIF4AIbinding domains of eIF4GI as well as for co-purification ability to these two domains. The E186 residue was found to be of significance for RNA-dependent ATPase activity for eIF4AI alone and in the presence of eIF4AI-binding domains of eIF4GI through point-mutation analysis. Furthermore, binding interactions between eIF4AI and eIF4GI domains were also significantly influenced by mutation of E186, as observed through co-purification assays. Thus, this residue appears to be of functional significance for eIF4A. Key words: DEAD-box family, helicase, superfamily 2, translation initiation factor, eukaryotic translation initiation factor 4A. Résumé : La famille de protéines a` boîte DExD (boîte DEAD) a été criblée afin de déterminer si des séquences spécifiques au facteur d’initiation de la traduction eucaryote 4A (eIF4A) était présentes dans les environs de la boîte DEAD. Un résidu glutamate unique a` eIF4A (E186 chez eIF4AI) a été identifié immédiatement après la séquence D-E-A-D chez eIF4AI, II et III, qui s’est avéré conservé de la levure a` l’humain. Des mutations ont été introduites a` l’intérieur de eIF4A recombinant exprimé chez Escherichia coli afin d’obtenir une sélection d’acides aminés alternatifs, et les protéines mutantes ont été étudiées afin de déterminer si une activité ATPase dépendante de l’ARN était présente. Les mutants ont aussi été étudiés afin de déterminer si des changements d’activité survenaient en présence des deux domaines de liaison de eIF4AI et eIF4GI, et vérifier la capacité de co-purification de ces deux domaines. Le résidu E186 s’est avéré important a` l’activité ATPase dépendante de l’ARN chez eIF4AI seul, ainsi qu’en présence des domaines de liaison de eIF4AI de eIF4GI, selon l’analyse de mutation ponctuelle. De plus, les interactions de liaison entre eIF4AI et les domaines de eIF4GI étaient aussi significativement influencées par la mutation de E186 tel qu’observé lors des tests de co-purification. Ainsi, ce résidu semble être d’une importance fonctionnelle importante pour eIF4A. [Traduit par la Rédaction] Mots-clés : famille a` boîte DEAD, hélicase, superfamille 2, facteur d’initiation de la traduction, facteur d’initiation de la traduction eucaryote 4A.

Introduction The DExD/H-box family of proteins is composed of DExD-box (DDX proteins) and DExH-box proteins (DHX proteins), where x is often Ala, that are necessary for a number of critical cellular processes ranging from transcription, splicing, protein synthesis, ribosome biogenesis, and RNA decay (Abdelhaleem et al. 2003; Cordin et al. 2006; Fairman-Williams et al. 2010; Linder and Jankowsky 2011; Rocak and Linder 2004). Nominally, DExD/H-box proteins share a number of highly conserved motifs that participate in RNA binding/unwinding (helicase activity) and ATP hydrolysis (ATPase activity). The DExD-box and the DExH-box families are clearly distinguished by the presence of an Asp or a His, respectively, in the fourth position of motif II. Additional distinct differences between the two families are found within the other motifs (Cordin et al. 2012; Rocak and Linder 2004). DExD/H-box proteins share a similar core structure that forms two RecA-like domains, and differences between family members

lie in additional protein domains/sequences beyond the core (Linder and Jankowsky 2011). The prototypical DExD-box member is the translation initiation factor eIF4A (eukaryotic translation initiation factor 4A), which consists of the two RecA-like domains and minimal extensions at the N- and C-termini (Andreou and Klostermeier 2013; Rogers et al. 2002). There are three isotypes of eIF4A in humans, eIF4AI (DDX2A) and eIF4AII (DDX2B) that are functionally indistinguishable (Nielsen and Trachsel 1988), and eIF4AIII (DDX48) (approximately 60% identical to I and II) that cannot substitute for eIF4AI/II in translation initiation and is a member of the exon-junction complex (EJC) (Ballut et al. 2005; Bono and Gehring 2011; Li et al. 1999). The eIF4AI and II isotypes (approximately 90% identical) are necessary for cap-dependent translation initiation (Hinnebusch and Lorsch 2012; Marintchev 2013; Pestova et al. 2007). ATP and RNA bind co-operatively to eIF4A, which undergoes a cycle of conformational changes associated with the hydrolysis of

Received 31 July 2013. Revision received 20 November 2013. Accepted 2 December 2013. K. Patel,* G.K. Shah,† S.S. Kommaraju, and W.-K. Low. Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John’s University, 8000 Utopia Parkway, Queens, NY 11439, USA. Corresponding author: Woon-Kai Low (e-mail: [email protected]). *Present address: Manus Biosynthesis, 790 Memorial Drive, Suite 102, Cambridge, MA 02139, USA. †Present address: Amneal Pharmaceuticals, 50 Horseblock Road, Yaphank, NY 11980, USA. Biochem. Cell Biol. 92: 33–42 (2014) dx.doi.org/10.1139/bcb-2013-0076

Published at www.nrcresearchpress.com/bcb on 4 December 2013.

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ATP and unwinding of RNA duplexes (Andreou and Klostermeier 2013; Lorsch and Herschlag 1998a, 1998b). In translation initiation, eIF4AI/II is a member of eIF4F that includes eIF4E (5=-cap binding protein) and eIF4G, a scaffolding protein involved in coordinating the interactions of eIF4F with the 43S pre-initiation complex (PIC) for loading of the small ribosome onto the 5=-end of mRNAs (Jackson et al. 2010; Parsyan et al. 2011). eIF4AI/II removes secondary structure within the 5=-UTRs (untranslated regions), and eIF4AI/II activity is regulated by contacts with eIF4G and two other proteins, eIF4B and eIF4H (Andreou and Klostermeier 2013; Grifo et al. 1984; Jackson et al. 2010; Parsyan et al. 2011; Pause et al. 1994; Richter-Cook et al. 1998). We have identified E186 within human eIF4AI immediately following the DEAD box that is also found in all three mammalian eIF4As and in the yeast homolog, tif1p, but is not found in any of the other human DDX proteins. We have investigated the functional significance of this conserved Glu through generation of recombinant proteins with mutation at this position to amino acids found in other human DDX proteins. Our results indicate that E186 has a significant role in determining the final functional activity of eIF4AI.

Materials and methods Protein expression vectors The N-terminally His-tagged 6XHis-eIF4AI WT expression vector has been described previously (Low et al. 2005, 2007). E186 mutations were created by site-directed mutagenesis of the WT expression vector using the QuikChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, USA) as described by the manufacturer. The primer sequences used to create each E186 mutation are given in the Supplementary data Table S1, and mutations were confirmed by DNA sequencing (data not shown).1 N-terminally His-tagged expression vectors for human eIF4GI (736-1115) and (1118-1600) cloned into the pET28b(+) vector (EMD Millipore, Billerica, USA) were kindly provided by Dr. Tatyana Pestova (SUNY Downstate Medical Center, Brooklyn, NY, USA). N-terminally GST-tagged versions of human eIF4GI fragments were created by PCR amplification of the respective eIF4GI regions using the primers given in Supplementary data Table S2.1 PCR primers contained restriction enzyme sites for XhoI and EcoRI to allow for in-frame cloning of PCR products into the pGEX-6P2 (GE Healthcare Bioscences, Pittsburgh, USA) expression vector. Resulting expression vectors were confirmed by DNA sequencing (data not shown). Protein expression and purification The eIF4AI WT and mutant proteins were purified as previously described (Low et al. 2005, 2007). After purification, buffer was exchanged to 20 mmol/L Tris-HCl, 100 mmol/L KCl, 0.1 mmol/L EDTA, 2 mmol/L DTT, 20% glycerol (v/v), pH 7.4 by dialysis and each protein was concentrated to minimum 1 mg/mL and stored at −80 °C. The same protocol was followed for purification of Histagged eIF4GI protein fragments. For GST-tagged eIF4GI protein fragments, protein expression was induced by addition of 1 mmol/L IPTG to exponential phase Escherichia coli BL21 (DE3) grown at 37 °C for 3 h. Cells were then harvested and lysed by sonication in 50 mmol/L Tris-HCl, 150 mmol/L NaCl, 2% Triton-X-100 (v/v), pH 7.5. Soluble protein was collected by centrifugation of cell debris, and supernatant was mixed with glutathione-S-agarose (Pierce Biotechnology, Rockford, USA) pre-equilibrated with 50 mmol/L TrisHCl pH 7.5. After washing of resin, bound protein was eluted with 50 mmol/L Tris-HCl, 10 mmol/L reduced glutathione pH 8.0 followed by concentration to minimum 1 mg/ml and dialysis into

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50 mmol/L Tris-HCl, 1 mmol/L EDTA, 1 mmol/L DTT, 20% glycerol (v/v) pH 7.5 and storage at −80 °C. ATPase assays For eIF4AI WT and mutant proteins ATPase assays were performed as previously described with minor modifications (Korneeva et al. 2005; Low et al. 2005, 2007). 20 ␮L reactions contained 0.5 ␮mol/L WT eIF4AI or mutant protein in buffer 20 mmol/L Tris-HCl, 0.5 mmol/L MgCl2, 100 mmol/L KCl, 2 mmol/L DTT, 0.1% Tween-20, 10% glycerol (v/v), and 1 mg/mL BSA, pH 7.5. Reactions were initiated by addition of poly(U) RNA (Sigma-Aldrich, St. Louis, USA) dissolved in H2O. Poly(U) RNA concentrations were determined as previously described (Lorsch and Herschlag 1998a) by calculating for 20 mer units. ATP (Sigma-Aldrich, St. Louis, USA) stock solution was prepared with equimolar MgCl2, then [␥-32P]-ATP, 3000 ␮Ci/mmol (PerkinElmer, Waltham, USA) was diluted 10-fold into 200 mmol/L Mg2+·ATP. Each protein was first tested in continuous assays under saturating ATP and RNA concentrations incubated at 37 °C. All active proteins were found to have initial velocity activity within the first 20 min, with less than 20% substrate consumed. (Supplementary data Fig. S1).1 Single timepoint assays (20 min.) were then performed for kinetic analysis using Michaelis–Menten steady-state approximation. Fraction ATP hydrolyzed was determined as previously described (Merrick and Sonenberg 1997) with minor modifications. Reactions were quenched by sequential addition of 500 ␮L of 20 mmol/L silicotungstic acid and 20 mmol/L sulfuric acid, then 1.2 mL of 1 mmol/L KPO4, then 500 ␮L of 5% ammonium molybdate in 4 M H2SO4, and finally 300 ␮L of 5% trichloroacetic acid/acetone (1:1). Released Pi was extracted using isobutylalchohol:benzene (1:1), and scintillation counting was performed. Fraction hydrolyzed was determined by comparing released extracted Pi to total radioactivity in the reaction mixture. For reactions with His-tagged eIF4GI fragments, 0.5 ␮mol/L eIF4AI WT protein or mutant protein was combined with indicated molar ratios of His-tagged eIF4GI protein fragments or eIF4GI storage buffer in the same buffer system as for eIF4AI proteins alone. Reaction time was 20 min, concentrations of ATP and poly(U) were saturating (1 mmol/L and 500 ␮mol/L respectively), and Pi was extracted and fraction hydrolyzed was determined as described above. Co-purification assay 500 ␮L reactions consisting of 5 ␮mol/L of eIF4AI WT or mutant protein and 1 ␮mol/L of either GST-tagged eIF4GI (736-1115) or (1118-1600), were combined with 10 ␮mol/L poly(U) RNA (12-mer oligo), and 100 ␮mol/L Mg2+·AMPPNP or 100 ␮mol/L Mg2+·ADP, in 20 mmol/L Tris-HCl, 0.5 mmol/L MgCl2, 100 mmol/L KCl, 2 mmol/L DTT, 0.1% Tween 20, and 5 mg/mL of BSA, pH 7.5. AMPPNP, and ADP (Sigma-Aldrich, St. Louis, USA) stock solutions with equimolar MgCl2 were prepared as described for ATP. Binding mixtures were incubated on ice for 40 min, and negative controls contained eIF4GI storage buffer only. Binding mixtures were then incubated with glutathione-S-agarose for 1 h at 4 °C with mixing followed by 3 washes with 1 mL of 1× binding buffer only. Captured proteins were then liberated by boiling the resin with SDS-PAGE loading buffer for 10 min. Purified proteins were visualized by SDS-PAGE followed by immunoblotting with anti-His tag and anti-GST tag antibodies (Rockland Immunochemicals, Gilbertsville, USA).

Results Alignment of DEAD box sequences in the DDX family The DExD-box family of proteins (DDX proteins) was examined for possible functionally significant residues conserved only in eIF4A (I, II, and III, DDX2A, DDX2B, and DDX48, respectively, un-

Supplementary data are available with the article through the journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/bcb-2013-0076. Published by NRC Research Press

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less otherwise stated, eIF4A throughout the remainder of the text will refer to all three isotypes, and when specific isotypes are discussed the numerical designations will be included) and not found in other DDX proteins. The UniProt database was manually searched for all human DDX protein sequences, and a manual alignment was performed for 38 DEAD-box sequences (see Fig. 1 and Supplementary data for further details of sequence alignment procedure).1 The majority of DDX proteins possessed an Arg residue immediately following the DEAD box, and comprised 50% of the protein sequences surveyed. Lys was the next most represented residue at approximately 18.4%. The next largest group (⬃18.4%) was uncharged residues at physiological pH (excluding Gln, see below), with Val being the most represented amino acid within this group. For all three eIF4As, the amino acid immediately following the DEAD box was a Glu residue. Furthermore, a Glu residue was found within tif1p, the yeast homolog of human eIF4AI. In eIF4AI, the positional number of the Glu is 186, and further discussion of this residue will refer to the numbering within eIF4AI (i.e., E186), although the corresponding amino acid is found at different positions within the different proteins (see Fig. 1). Another small group composed of only DDX21, and DDX50 possessed the structurally similar residue Gln at this position, although these two proteins possess atypical DEAD-box sequences of D-E-V-D where Val replaces the Ala found in the third position of motif II. Nevertheless, due to the structural similarities between Glu and Gln amino acids, the decision was made to separate DDX21 and DDX50 from the group containing other uncharged residues at physiological pH. As Glu was only found in eIF4A and was conserved from yeast to mammals, this raised the possibility that Glu at this position has some functional significance. No immediately observable correlation within the grouping presented here and the general functions of each of the DDX proteins, as listed in the RNA helicase database (http://www.rnahelicase.org/), were immediately apparent. Mutational analysis of E186 in eIF4AI Investigation of the functional significance of the conservation of E186 was begun by generation of point mutations in an N-terminally His-tagged recombinant protein of human eIF4AI produced in E. coli that has been previously reported on (Low et al. 2007, 2005). The unchanged amino acid sequence will be referred to as WT although the recombinant protein possessed a nonnative sequence at the N terminus comprising the His-tag. Point mutations were designed to replace E186 with either Arg (E186R), Lys (E186K), Gln (E186Q), or Ala (E186A). All recombinant proteins also possessed the same nonnative N-terminal His-tag as WT. Arg and Lys residues were selected for investigation due to their high representation within the DDX family, Gln was chosen because of its structural similarity to Glu, and Ala was chosen as a standard method of side chain elimination. All recombinant proteins were expressed in E. coli and purified to greater than 90% purity (data not shown), then assayed for ATPase activity. For each protein, timecourse analysis of RNA-dependent ATPase activity was performed over 1 h (Supplementary data Fig. S1)1 under saturating ATP concentrations (1 mmol/L ATP) and reactions were initiated by addition of saturating poly(U) RNA (500 ␮mol/L, see experimental procedures for description of RNA concentration determination). No proteins demonstrated ATPase activity in the absence of RNA addition, and only WT, E186Q, and E186K demonstrated RNA-dependent ATPase activity, although the activity of E186K was extremely low (Supplementary data Fig. S1 and Fig. 2).1 The E186A and E186R mutations only displayed detectable hydrolysis at the 60 min timepoint, but the readings were extremely low and difficult to distinguish from background (Supplementary data Fig. S1).1 Based on the timecourse of activity, WT, E186Q, and E186K were determined to have linear activity to the 20 min timepoint with

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Fig. 1. Glutamate immediately following the DEAD box of motif II is unique to eIF4A in the DDX family of proteins. Manual alignment of the protein sequence surrounding the DEAD box of human DDX proteins. Human DDX proteins were manually surveyed within the UniProt database, and the protein sequences were manually aligned in the region surrounding the DEAD box (underlined text) and are shown with the assigned DDX nomenclature. The amino acid residue immediately following the DEAD motif is highlighted (bold text), and the proteins were grouped according to the identity of this amino acid. For comparison, the yeast homolog of human eIF4AI (DDX2A), TIF1, was also included. The amino acid position within each protein that corresponded to E186 in human eIF4AI (DDX2A) is given in parenthesis to the right. To the left in parentheses are the percentage representations of each grouping within this survey.

less than 20% substrate utilization, and more detailed fixedtimepoint Michaelis–Menten kinetic analysis of activity was performed (see Fig. 2). First, ATPase activity for the three proteins was investigated under conditions of saturating poly(U) RNA with varying ATP concentration. As has been previously reported (see Cordin et al. 2006 for review), low overall activity was observed for WT, with a kcat of approximately 3.6 min−1, a Km of approximately 405 ␮mol/L, and a catalytic efficiency of 1.5 × 102 (mol/L)−1 s−1 (Figs. 1A and 1C). Aside from the higher Km, these values were in Published by NRC Research Press

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Fig. 2. Mutation of E186 to glutamine improves catalytic efficiency in human eIF4AI. RNA-dependent ATPase activity of N-terminally His-tagged eIF4AI or E186 mutations. All measurements were taken under initial velocities and quenched after 20 min. Each data point represents the mean of 3–5 independent assays with error bars representing ± 1 SEM. No activity was observed in all instances in the absence of RNA (data not shown). (A) Conditions of varying ATP concentration with saturating poly(U) RNA (500 ␮mol/L). (B) Conditions of varying RNA concentration and saturating ATP (1 mmol/L). (C) Kinetic parameters as determined under the steady-state approximation. The kcat and kact are presented with the atypical unit of min−1 due to the low activity.

good agreement with our previously reported results for this protein (Low et al. 2005, 2007). The differences in the Km may have been due to differences in the assay procedure in this report compared to previous reports, although higher Km values in the

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300–400 ␮mol/L range for eIF4AI have been reported in the past (Blum et al. 1992; Rogers et al. 1999, 2001a, 2001b). For E186Q, turnover rate was very similar to WT, but a significant decrease in Km was observed (102 ␮mol/L for E186Q versus 405 ␮mol/L for WT, Fig. 1A & 1C). This decrease in Km lead to an overall increase in catalytic efficiency of approximately 3.8-fold (1.5 × 102 (mol/L)−1 s−1 for WT versus 5.7 × 102 (mol/L)−1 s−1 for E186Q). Although the activity of E186K was extremely low, the same analysis was performed for this mutation, however, due to the low activity and thus low reliability, kinetic parameters are not reported although the activity of E186K is presented (Fig. 2A). Next, the ATPase activities of WT, E186Q, and E186K were examined under saturating ATP concentrations (1 mmol/L) and varying poly(U) RNA concentration (Fig. 2B). Under these conditions, E186Q demonstrated slightly greater turnover rate (3.6 min−1 for E186Q versus 3.0 min−1 for WT), and slightly higher Km (30.3 ␮mol/L for E186Q versus 18.6 ␮mol/L for WT). Overall catalytic efficiency of E186Q under these conditions was slightly greater than WT (19.8 × 102 (mol/L)−1 s−1 for E186Q versus 26.8 × 102 for WT). Again, the low activity of E186K is presented (Fig. 2B), but kinetic parameters were not determined. Stimulation of ATPase activity by eIF4G The activity of free eIF4AI is expected to be negligible for translation initiation (Marintchev 2013), and in vivo function of eIF4AI in translation initiation minimally requires interaction with eIF4G that stimulates eIF4AI activity and also likely further requires stimulation by eIF4B and eIF4H (Andreou and Klostermeier 2013). Within full-length eIf4GI (1–1600 amino acids) there are two eIF4AI-binding domains, one within the central domain and one within the C-terminal third of the protein. Previous reports have demonstrated that the central domain of eIF4GI stimulates eIF4AI activity, whereas the C-terminal domain appears to modulate the activity (see Discussion for further detail) (Andreou and Klostermeier 2013). Thus, to examine the activity of our eIF4AI mutations in the presence of the eIF4G domains, two N-terminally His-tagged recombinant proteins were produced, 4GI (736–1115) and 4GI (1118–1600), where the numbering following 4GI denotes the amino acid positional numbering from full-length eIF4GI and were the amino acids from eIF4GI included in each protein. The 4GI recombinant proteins were purified to ⬃90% purity and did not demonstrate any detectable ATP hydrolysis activity even in the presence of added poly(U) RNA (data not shown). The ATPase activity of WT was assayed under saturating ATP (1 mmol/L) and saturating poly(U) RNA concentrations (500 ␮mol/L) for 20 min in the presence of 4GI (736–1115). The activity was also tested under varying molar ratios of the two proteins, 1:1, 1:2, and 1:3 WT:4GI (736–1115). WT alone was re-assayed as the buffer and conditions were slightly different than the assays reported above. Statistically significant increases in activity were observed in the presence of 1:1 and 1:2 molar ratios of WT:4GI (736–1115), however no change in activity was observed when a 3-fold excess of 4GI (736–1115) was present in the assay (Supplementary data Fig. S2A).1 A similar assay was performed replacing 4GI (736–1115) with 4GI (1118–1600). In this instance, no statistically significant change in ATPase activity was observed at 1:1 and 1:2 molar ratios of WT:4GI (1118–1600), and a decrease in activity was observed at 1:3 molar ratio (Supplementary data Fig. S2B).1 The 1:1 ratio of WT:4GI (736–1115) resulted in only a modest ⬃2-fold increase in activity. This is in contrast to other reports such as in Ozes et al. 2011, where an ⬃10-fold increase in activity was observed. This difference is likely due to the differences in the truncations of eIF4GI used. Given the results presented in Fig. S2 in the Supplementary data,1 a molar ratio of 1:1 was selected for further ATPase activity studies, where WT was replaced with the mutated forms of eIF4AI. E186R and E186A did not demonstrate appreciable ATPase activity in isolation, and the presence of either 4GI (736–1115) or 4GI (1118–1600) did not stimulate these proteins to any detectable level (data not Published by NRC Research Press

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shown). The stimulatory activity of 4GI (736–1115) and the unchanged activity in the presence of 4GI (1118–1600) on WT was also reconfirmed (Figs. 3A and 3B, respectively). WT alone demonstrated activity of 2.1 ± 0.4 ␮mol/L of Pi/mol enzyme/min, which was stimulated nearly 2-fold in the presence of the 4GI (736–1115). No significant change in activity was observed in the presence of 4GI (1118–1600). Next, the impact of the eIF4GI fragments on E186Q was examined. E186Q again demonstrated higher activity than WT (3.8 ± 0.3 ␮mol/L of Pi/mol enzyme/min). In the presence of both eIF4GI fragments, the ATPase activity of E186Q was drastically reduced to below 50% activity of E186Q alone (Figs. 3C and 3D). E186K was also investigated even though it presented extremely low activity of 0.8 ± 0.3 ␮mol/L of Pi/mol enzyme/min (also see Figs. 2A and 2B). For E186K, no statistically significant change in activity was observed, although the mean activities for E186K were lower in the presence of the 4GI fragments (Figs. 3E and 3F). Co-purification with eIF4GI Changes in enzymatic activity in the presence of the eIF4GI recombinant protein fragments suggested that E186Q still physically interacted with the eIF4A-binding domains within eIF4GI. To assess the impact of the mutations at position E186, GST-tagged forms equivalent to the His-tagged fragments of eIF4GI were produced in, and purified to ⬃80% purity from E. coli (data not shown). Each of the eIF4AI mutants or the WT protein were incubated with each of the two GST-tagged eIF4GI recombinant proteins and then assessed for their co-purification by glutathione-agarose resin. Although a 1:1 ratio of recombinant proteins was determined to be the optimum ratio in the ATPase assays above, a 5:1 ratio of Histagged eIF4A and mutant recombinant proteins to recombinant GST-tag eIF4GI proteins was used to ensure maximum binding under the co-purification conditions. While co-affinity purification is not a truly quantitative binding assay, the amount of protein co-captured should indirectly reflect the strength of interaction under the binding conditions. The amount of mutant or WT protein that was co-purified was determined by immunoblotting with anti-His primary antibodies and normalized to the amount of GST-tagged eIF4GI recombinant protein that was purified as determined by immunoblotting with anti-GST antibodies. Each experiment was carried out in triplicate, with one representative co-purification assay shown in Figs. 4 and 5 (panels A and B), and mean values for the amount of co-captured protein was determined. For each of the mutant proteins, the difference in amount co-captured was compared to WT (Figs. 4C and 5C). Copurification assays were performed to mimic interactions during the catalytic cycle of eIF4AI by including the non-hydrolyzable ATP analog AMP-PNP and an 12-mer of poly(U) (panel A in Figs. 4 and 5) to mimic the “pre-catalytic state”, or ADP and the 12-mer of poly(U) (panel B in Figs. 4 and 5) to mimic the “post-catalytic state”. A short oligomer of poly(U) was utilized to minimize through-RNA protein–protein interactions as both eIF4AI, and the central domain of eIF4GI possess RNA-binding activity. Under these assay conditions, no detectable interactions were observed for all proteins without the presence of eIF4AI substrates (nucleotide and RNA) (data not shown). For binding to the central domain of eIF4GI under conditions mimicking the pre-catalytic state, mutation of E186 to Gln (E186Q) demonstrated approximately 2-fold greater co-purification compared to WT (Fig. 4A, lane 3 versus lane 1). Greater co-purification was also observed for E186Q under conditions mimicking the post-catalytic state with an observed 1.4-fold increase (Fig. 4B, lane 3 versus lane 1). Under pre-catalytic mimicking conditions, E186A demonstrated very little co-purification to the eIF4GI central domain, and no co-purification was detected under post-catalytic conditions (Figs. 4A and 4B, lanes 5 versus lane 1). For E186K and E186R, less than 50% co-purification to the central domain of eIF4GI was observed compared to WT for pre-catalytic mimicking conditions (Fig. 4A, lanes 7 and 9, respectively, versus lane 1).

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Fig. 3. Mutation of E186 in eIF4AI to glutamine or lysine perturbs the effects of the eIF4AI-binding domains of eIF4GI on eIF4AI ATPase activity. RNA-dependent ATPase activity of WT, or indicated eIF4AI mutations alone or in the presence of indicated eIF4AI-binding domains of eIF4GI. ATPase activity was observed in reactions that contained 0.5 ␮mol/L N-terminally His-tagged eIF4AI (abbreviated 4AI) WT protein, mutation E186Q, or mutation E186K with N-terminally His-tagged recombinant eIF4GI central eIF4AI-binding domain (736–1115) or C-terminal eIF4A-binding domain (1118–1600) in a 1:1 molar ratio. Reactions were performed under saturating ATP and poly(U) concentrations (1 mmol/L and 500 ␮mol/L respectively). No activity was observed in the absence of RNA addition, and eIF4GI recombinant proteins alone did not demonstrate any activity (data not shown). For comparison, the activity of WT or mutations alone was considered 100%, and each data point was collected from 3–5 independent assays with mean values presented with +1 SEM. Statistically significant differences (*) were determined using student’s t test (p value < 0.05). (A–F) Protein components in each reaction as indicated. See Supplementary data Fig. S3 for absolute activity values.1

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Fig. 4. Mutation of E186 in eIF4AI perturbs the binding interaction between eIF4AI and the central stimulatory domain of eIF4GI. 1 ␮mol/L of N-terminal GST-tagged central eIF4A-binding domain of eIF4GI (736–1115) was incubated with indicated eIF4AI WT, or mutated recombinant proteins in a 1:5 molar ratio on ice for 40 min in the presence of (A) 100 ␮mol/L AMP-PNP and 10 ␮mol/L 12-mer poly(U) or (B) 100 ␮mol/L ADP and 10 ␮mol/L 12-mer poly(U) with 5 mg/mL BSA to mimic the pre-catalytic state (A) or post-catalytic state (B). GST-tagged eIF4GI (736–1115) and bound proteins were captured using glutathione-S-agarose and visualized by SDS-PAGE followed by immunoblotting detection using anti-GST tag and anti-His tag antibodies. Binding assays were performed in 3 independent assays and representative assays are shown in (A) and (B). Amount of eIF4AI WT or mutations co-purified were normalized to amount of GST-tagged eIF4GI (736–1115) captured. (C) Mean fold-changes with ±1 SEM in amount of mutant eIF4AI proteins co-captured compared to WT in each assay. Statistically significant differences (*) were determined using student’s t test (p value < 0.05). N.A., not applicable as protein was not detectable by immunoblotting.

Under post-catalytic mimicking conditions, no detectable copurification of the E186K mutation to the central domain was observed and less than 50% co-purification compared to WT was observed for E186R (Fig. 4B, lanes 7 and 9, respectively, versus lane 1).

For interactions with the C-terminal portion of eIF4GI, the E186Q mutant demonstrated reduced co-purification (less than 50%) when compared to WT under pre-catalytic mimicking conditions (Fig. 5A, lane 3 versus lane 1) and roughly equal binding to WT under postcatalytic mimicking conditions (Fig. 5B, lane 3 versus lane 1). For Published by NRC Research Press

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Fig. 5. Mutation of E186 in eIF4AI perturbs the binding interaction between eIF4AI and the C-terminal modulatory domain of eIF4GI. 1 ␮mol/L of N-terminally GST-tagged C-terminal eIF4A-binding domain of eIF4GI (1118–1600) was incubated with indicated eIF4AI WT, or mutated recombinant proteins in a 1:5 molar ratio on ice for 40 min in the presence of (A) 100 ␮mol/L AMP-PNP and 10 ␮mol/L 12-mer poly(U) or (B) 100 ␮mol/L ADP and 10 ␮mol/L 12-mer poly(U) with 5 mg/mL BSA to mimic the pre-catalytic state (A) or post-catalytic state (B). GST-tagged eIF4GI (1118–1600) and bound proteins were captured using glutathione-S-agarose and visualized by SDS-PAGE followed by immunoblotting detection using anti-GST tag and anti-His tag antibodies. Binding assays were performed in 3 independent assays and representative assays are shown in (A) and (B). Amount of eIF4AI WT or mutations co-purified were normalized to amount of GST-tagged eIF4GI (1118–1600) captured. (C) Mean fold-changes with ±1 SEM in amount of mutant eIF4AI proteins co-captured compared to WT in each assay. Statistically significant differences (*) were determined using student’s t test (p value < 0.05)

E186A, co-purification was less than 50% compared to WT under pre-catalytic mimicking conditions (Fig. 5A, lane 5 versus lane 1) and roughly 70% under post-catalytic mimicking conditions (Fig. 5B, lane 5 versus lane 1). The E186K mutation demonstrated approximately 60% co-purification under pre-catalytic mimicking conditions (Fig. 5A, lane 7 versus lane 1) and equivalent co-purification under post-catalytic mimicking conditions (Fig. 5B, lane 7 versus lane1)

when compared to WT, and the E186R mutation demonstrated approximately equal co-purification to WT under both conditions tested (Figs. 5A and 5B, lanes 9 versus lanes 1). It should also be noted that inclusion of E186R consistently reduced the amount of GST-tagged eIF4GI (1118–1600) that was purified using glutathione-S-agarose suggesting that binding to E186R interfered with the interaction with the resin. Published by NRC Research Press

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Discussion Given the fact that DDX proteins participate in a wide variety of cellular functions, it is postulated that the different DDX proteins acquire their specific functions through further protein domain additions at the N- and C-termini of the core sequence. Here we have demonstrated that the Glu residue conserved from yeast to mammals that immediately follows the DEAD box motif was critical for normal eIF4AI RNA-dependent ATPase activity and may be of importance for its unique binding interactions with the eIF4Abinding domains of eIF4GI. These results implicate the E186 position as a critical residue for proper functioning of the eIF4F complex during cap-dependent translation initiation. Importantly, glutamate was not found in any other DDX proteins known to date aside from the eIF4As. Loss of the side chain at E186 by replacement with Ala or replacement with a much larger and oppositely charged side chain in the form of Arg resulted in no detectable RNA-dependent ATPase activity. As there was no detectable ATPase activity, we cannot infer from kinetic data information concerning changes in binding affinities for ATP or RNA. These two mutations, E186A and E186R, may have resulted in proteins unable to bind to ATP and (or) RNA. For E186A, it seems unlikely that the mutant protein lost all ability to bind nucleotide (ATP or ADP), as in the co-purification assays with GST-tagged recombinant eIF4GI domains, the copurification levels were not identical for pull-downs using AMPPNP or ADP, demonstrating nucleotide-specific effects. As all co-purification assays contained equivalent amounts of the identical poly(U) oligo, and because eIF4GI itself is capable of binding to RNA, similar conclusions cannot be made for RNA. For E186R, very little variation was observed between the two co-purification conditions tested, thus loss of substrate binding ability is a likely possibility for E186R. Although further assays were not pursued with E186A and E186R due to their lack of activity, it may be of interest to investigate substrate binding of the mutations directly at a future date. The fact that RNA-dependent ATPase activity was retained in the E186Q protein compared with the other mutations suggests that there is a steric requirement at this location within the protein. The DDX proteins, including eIF4A, are proposed to cycle through “open” and “closed” conformations that are promoted by cooperative binding of ATP and RNA during their catalytic cycle, with specific spatial arrangements between the N- and C-terminal RecA-like domains necessary for activity (Andreou and Klostermeier 2013; Lorsch and Herschlag 1998a, 1998b). Thus, it is possible that mutations at E186 other than to the isosteric Gln negatively impacted the cycle of conformational changes necessary for activity. In the E186Q mutation, catalytic efficiency of the enzyme imthat may suggest proved due mostly to a 4-fold decrease of KATP m that in the presence of Glu (WT) negative charge–charge repulsion reduces ATP binding affinity. However, structural information of eIF4A and its homologs suggests that the 186 position side-chain is not in the vicinity of the ATP binding pocket, and the proposed charged-charge repulsion may be with another region or residue within the protein itself. Removal of the negatively charged Glu and replacement with the isosteric Gln may have made the ATPbound conformation of the protein more accessible. This scenario may be more likely, given the fact that the Arg in the corresponding position to E186 of eIF4AI in the crystal structure Vasa was found nearer to the RNA binding region than the ATP-binding pocket (Sengoku et al. 2006), which was also observed for the corresponding Glu in eIF4AIII when co-crystallized in the EJC (Andersen et al. 2006; Bono et al. 2006). In fact, the initial prediction was that removal of the negative charge by replacement of Glu with Gln would result in a significant decrease in the KRNA m , but a less than 2-fold increase was observed. It is also important to note that while replacement with the oppositely charged Lys did not fully abrogate activity, the ATPase activity was significantly

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decreased, and activity was not stimulated in the presence of the stimulatory central domain of eIF4GI. Thus, the effects of the residue at position 186 on activity cannot be solely electrostatic, and steric factors are may also involved. In translation initiation, eIF4AI/II functions as a component of eIF4F by directly interacting with eIF4G. In its longest form of 1600 aa, there are two eIF4AI binding domains in eIF4GI located centrally and within the C-terminus domains. The central domain stimulates eIF4A activity, while the C-terminal binding domain appears to play a regulatory role is not present in yeast and is not essential for stimulation of cap-dependent translation initiation (Imataka and Sonenberg 1997; Korneeva et al. 2001, 2005; Lamphear et al. 1995; Morino et al. 2000; Yang et al. 2004). Thus, binding interactions with eIF4G promote and regulate the cycle of conformational changes that occur within eIF4A during its activity. A number of structural studies have investigated the stimulation and regulation of eIF4AI by eIF4G within eIF4F (Hilbert et al. 2011; Marintchev et al. 2009; Oberer et al. 2005; Schutz et al. 2008) While differences exist in the models proposed from these studies, all are in agreement that direct binding of eIF4AI to the central eIF4AI-binding domain within eIF4G promotes conformational changes (a more “closed” or “half open” conformation) within eIF4AI that stimulates eIF4AI activity. Furthermore, when present the C-terminal domain also makes direct contacts with eIF4AI to regulate activity. In these structural studies, the eIF4AI surface containing E186 was not part of direct contact sites with eIF4G as the DEAD box was present on the distal side of eIF4AI to eIF4G. Thus, the changes in co-purification between eIF4AI and the eIF4GI fragments that were observed here upon mutation of E186 were likely due to indirect effects leading to changes in protein conformations that altered the binding surface and not due to direct disruption of contacts between the two proteins. This interpretation is supported by the evidence that DDX3, a DEAD-box protein with an Arg immediately following the D-E-A-D sequence, is also capable of interacting with eIF4G (Soto-Rifo et al. 2012, 2013). Interestingly, the E186Q mutation that resulted in enhanced ATPase activity, but lower ATPase activity in the presence of the middle stimulatory domain of eIF4GI, also demonstrated increased co-purification in both the pre-catalytic and post-catalytic mimicking binding assays. This result may suggest that removal of the negative charge at E186 disrupts the “conformational guidance mechanism” of eIF4G that stimulates the rate-limiting step of phosphate release (Hilbert et al. 2011), or due to the mutation, eIF4A binding to eIF4G increases in affinity and becomes “trapped” in a more closed conformation and cannot complete the conformational cycling necessary for phosphate release and “resetting” of the eIF4AI conformation for the next hydrolysis event. Regardless of the mechanism involved, the changes in co-purification for all E186 mutations to both eIF4GI domains and the changes in ATPase activities strongly suggest that the 186 position may play a significant role in the regulation of eIF4AI activity by eIF4G within eIF4F during translation initiation. Furthermore, the changes in co-purification upon mutation to the other amino acids tested may suggest that in these proteins, steric changes away from Glu/Gln caused the proteins to adopt conformations, or prefer conformations, that were less optimal for eIF4G interaction. Only in one instance for the mutations other than E186Q was copurification approximately equivalent to WT, and that was for E186K to the C-terminal region under post-catalytic conditions. Thus, mutation of E186 to Lys may have promoted a more “open” conformation associated with product release, which would be in agreement with the lower ATPase activity of E186K and the lack of observed interaction with the central eIF4G domain. Caution must be employed, and full conclusions from the work presented here are lacking, as only minimal fragments of eIF4GI were investigated and because of the assumption that the co-affinity purification of proteins is indicative of binding affinity. The implications of Published by NRC Research Press

Patel et al.

our results within the larger context of full-length eIF4GI and the numerous other translation initiation factors should be further investigated. More in-depth studies will be necessary to investigate the impact of E186 on eIF4A activity within eIF4F, such as direct quantitative binding assays. Although we have made attempts here to assess changes in binding affinity, co-affinity purification does not directly quantify changes in binding affinity. Another limitation of the studies presented here is that the truncated forms of eIF4GI used here are most likely not fully indicative of the activities and effects that would be observed in the presence of full-length eIF4GI. Furthermore, although single-point mutations typically do not cause drastic alterations in protein structure, based on the results presented here, abolishment of the structural integrity of the eIF4A protein fold cannot be ruled out for the mutant proteins lacking in RNA-dependent ATPase activity. Outside of eIF4F, the activity eIF4AI has also been demonstrated to be simulated by direct association with eIF4B and eIF4H. Both eIF4B and eIF4H directly interact in a mutually exclusive manner with eIF4AI at overlapping sites (Andreou and Klostermeier 2013; Feng et al. 2005; Grifo et al. 1984; Marintchev et al. 2009; Rogers et al. 2001b, Rozovsky et al. 2008). Thus, further studies are warranted to gain a full mechanistic understanding of the role of E186 in eIF4A. Furthermore, the E186 position may also have importance for the interactions with eIF4B/H, as the region where the DEAD box is found in eIF4AI has been implicated in direct binding to eIF4B and eIF4H (Rozovsky et al. 2008). Thus, interesting future studies would be to investigate the importance, if any, of E186 in the stimulation of eIF4AI activity by eIF4B/H. The eIF4AIII isotype is distinct from I and II, as it does not participate in translation initiation, but is the “RNA clamp” that locks the EJC to RNA. Where eIF4AI/II interact with eIF4G, eIF4B, and eIF4H, eIF4AIII interacts with Magoh, Y14, and Barentsz (BTZ) (Ballut et al. 2005; Bono and Gehring 2011; Li et al. 1999). As a Glu residue immediately follows the DEAD box in eIF4AIII, it would also be interesting to determine if there is similar importance in eIF4AIII for this residue as well, in terms of activity and interaction within its protein network. Taken together, the results presented here may suggest that the Glu at position 186 in eIF4AI/II may be a distinguishing feature that determines the specificity of eIF4AI/II for translation initiation. Although a Glu is also found in eIF4AIII, other sequence differences may cause eIF4AIII incapable for substituting for eIF4AI/II in translation initation. Finally, the DExHbox protein DHX29, which has been found to be necessary along with eIF4F for efficient scanning of mRNAs with highly structured 5=-UTRs (Pisareva et al. 2008), also features a Glu residue immediately following motif II (DEVHE, aa 702–706). Obviously no inferences about the potential importance of E706 in DHX29 can be made purely on this observation, but it may suggest that further investigation of this amino acid position in DHX29 and (or) other DDX/DDH should also be pursued. In summary, the Glu residue immediately following the DEAD box within eIF4A proteins very likely has significant importance for the biological function of these proteins.

Acknowledgements The authors wish to thank Dr. Tatyana Pestova for provision of the original eIF4GI expression constructs. The work presented here was funded by St. John’s University.

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