Structure of eIF4E in Complex with an eIF4GPeptide Supports a Universal Bipartite Binding Modefor Protein Translation1[OPEN]

Manuel Miras,a Verónica Truniger,a Cristina Silva,b Núria Verdaguer,b Miguel A. Aranda,a,2 andJordi Querol-Audíb,2

aCentro de Edafología y Biología Aplicada del Segura (CEBAS), Consejo Superior de InvestigacionesCientíficas (CSIC), 30100 Espinardo, Murcia, SpainbInstitut de Biologia Molecular de Barcelona/CSIC, Parc Científic de Barcelona, 08028 Barcelona, Spain

ORCID ID: 0000-0002-0828-973X (M.A.A.).

The association-dissociation of the cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) with eIF4G is a keycontrol step in eukaryotic translation. The paradigm on the eIF4E-eIF4G interaction states that eIF4G binds to the dorsal surfaceof eIF4E through a single canonical alpha-helical motif, while metazoan eIF4E-binding proteins (m4E-BPs) advantageouslycompete against eIF4G via bimodal interactions involving this canonical motif and a second noncanonical motif of the eIF4Esurface. Metazoan eIF4Gs share this extended binding interface with m4E-BPs, with significant implications on the understandingof translation regulation and the design of therapeutic molecules. Here we show the high-resolution structure of melon (Cucumismelo) eIF4E in complex with a melon eIF4G peptide and propose the first eIF4E-eIF4G structural model for plants. Our structuraldata together with functional analyses demonstrate that plant eIF4G binds to eIF4E through both the canonical and noncanonicalmotifs, similarly to metazoan eIF4E-eIF4G complexes. As in the case of metazoan eIF4E-eIF4G, this may have very importantpractical implications, as plant eIF4E-eIF4G is also involved in a significant number of plant diseases. In light of our results, auniversal eukaryotic bipartite mode of binding to eIF4E is proposed.

The initiation of protein synthesis is a key control andhighly regulated step in eukaryotic gene expression(Sonenberg and Hinnebusch, 2009). Translation ini-tiation leads to the assembly of the large (60S) andsmall (40S) ribosomal subunits into an active 80S ribo-some that is able to locate the correct start codon ofthe mRNA molecule. This is facilitated by the coordi-nated actions of at least 12 protein initiation factors

(Browning, 2004; Jackson et al., 2010; Hinnebusch, 2011;Hinnebusch and Lorsch, 2012; Browning and Bailey-Serres, 2015). In cap-dependent translation, binding ofthe eukaryotic translation initiation factor 4F (eIF4F) tothe 7-methyl guanosine cap (m7G cap) at the 59 end ofmRNA drives the attachment of the mRNA to the ri-bosomal 43S preinitiation complex. In mammals, eIF4Fis built by three proteins: the mRNA 59 cap bindingprotein eIF4E, the RNA helicase eIF4A, and the scaf-folding protein eIF4G, which contains binding domainsfor eIF4E, eIF4A, eIF3, and poly(A)-binding protein(PABP; Marcotrigiano et al., 1997; Pestova et al., 2001;Gross et al., 2003). In plants, purified eIF4F hetero-dimers contain eIF4E and eIF4G (Hinnebusch andLorsch, 2012); plant eIF4A appears to be loosely as-sociated and is therefore easily lost during purifica-tion (Lax et al., 1986). EIF4G associates with eIF3,recruiting the 40S subunit of the ribosome and initi-ating scanning of the mRNA in the 59 to 39 direction(Jackson et al., 2010; Park et al., 2011). EIF4G addi-tionally binds simultaneously to eIF4E and PABP, thelatter being able to interact with the mRNA poly(A)tail (Aitken and Lorsch, 2012; Browning and Bailey-Serres, 2015), resulting in a transient circularizationof the mRNA.

Higher plants contain an additional isoform of theeIF4F complex, eIFiso4F (Lax et al., 1986; Browning andBailey-Serres, 2015). Plant eIF4E and eIFiso4E shareapproximately 50% amino acid identity, while eIFiso4G

1 Work in Murcia was financially supported by grants AGL2015-65838 and PCIN-2013-043 (MINECO, Spain-FEDER). Work inBarcelona was supported by grants BIO2014-54588-P (MINECO,Spain-FEDER) and Maria de Maeztu Unit of Excellence MDM-2014-0435. X-ray data were collected at ALBA-CELLS (beamline XALOC),Cerdanyola del Valles, Barcelona, Spain, with the collaboration ofALBA staff and at ESRF (Grenoble, France). Financial support wasalso provided by ALBA and ESRF.

2 Address correspondence to jquerolaudi@gmail.com andm.aranda@cebas.csic.es.

The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) are:Miguel A. Aranda (m.aranda@cebas.csic.es) and Jordi Querol-Audí(jquerolaudi@gmail.com).

M.M., J.Q.-A., V.T., and C.S. performed the experiments; J.Q.-A.,M.M., and V.T. analyzed the data; M.A.A., M.M., and J.Q.-A. con-ceived the study; J.Q.-A., M.M., M.A.A., and N.V. wrote the manu-script; all authors read and approved the final manuscript.

[OPEN] Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.17.00193

1476 Plant Physiology�, July 2017, Vol. 174, pp. 1476–1491, www.plantphysiol.org � 2017 American Society of Plant Biologists. All Rights Reserved. www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from

Copyright © 2017 American Society of Plant Biologists. All rights reserved.

differs from eIF4G in having a truncated N terminus,but shares similar binding domains for eIF4E, eIF3, andeIF4A. EIF4G interacts with eIF4E through a highlyconserved Y(X)4Lf amino acid sequence, where X isvariable and f is hydrophobic, known as the canonical(C) binding motif (Mader et al., 1995; Marcotrigianoet al., 1999; Gross et al., 2003). The C motif is also con-served in the plant-specific eIFiso4G subunit. AlthougheIFiso4G can form mixed complexes with eIF4E thatretain activity in vitro, dissociation constants showedmolecular specificity, suggesting a differential role ofthe eIFiso4F complex in translation initiation (Gallieand Browning, 2001; Mayberry et al., 2011). MetazoaneIF4E-binding proteins (m4E-BPs) also contain the Cmotif (Mader et al., 1995; Marcotrigiano et al., 1999) andinhibit translation initiation by competing for the samebinding site on the eIF4E surface, thus blocking theassembly of active translation eIF4F complexes (Maderet al., 1995; Matsuo et al., 1997; Marcotrigiano et al.,1999; Gross et al., 2003). In addition to the Cmotif, m4E-BPs also contain a downstream noncanonical (NC)domain that forms a loop and binds to a highly con-served hydrophobic lateral surface of eIF4E (Mizunoet al., 2008; Gosselin et al., 2011; Paku et al., 2012;Lukhele et al., 2013; Igreja et al., 2014; Peter et al., 2015a,2015b). An elbow loop downstream of the C motif ofm4E-BPs induces the bending of the peptide backbone,thus allowing the NC loop to reach and contact thelateral hydrophobic pocket of eIF4E (Kinkelin et al.,2012; Peter et al., 2015a, 2015b). In plants, only lipoxy-genase 2 (LOX2) and the beta subunit of the nascentpolypeptide-associated complex (BTF3) have beenidentified as putative eIF4E interacting proteins (Freireet al., 2000; Freire, 2005), and very little is known abouttheir association with eIF4E.The NMR structure of the yeast eIF4E-eIF4G393-490

complex has revealed that the C-terminal regiondownstream of the C motif folds back toward theflexible N-terminal tail of eIF4E, not interacting withthe lateral surface of eIF4E (Gross et al., 2003). Since thelateral interaction of the NCmotif seemed to occur onlyin m4E-BPs, it was proposed to be the reason for theknown advantage of m4E-BPs over eIF4G in bindingeIF4E, leading to translation repression (Paku et al.,2012; Lukhele et al., 2013; Igreja et al., 2014). However,the very recently solved crystal structure of Homo sa-piens and Drosophila melanogaster eIF4E-eIF4G peptidecomplexes has revealed that eIF4G also binds eIF4Ethrough both C andNCmotifs, coming into contact withthe same dorsal and lateral eIF4E surfaces, respectively,as m4E-BPs (Grüner et al., 2016). Additionally, Grüneret al. (2016) have shown that the known competitiveadvantage of m4E-BPs over eIF4G is provided by theamino acid composition of the linker and theNCbindingmotif.Here we present high-resolution structural data of

melon (Cucumis melo) eIF4E (Cm eIF4E) in complexwitha Cm eIF4G peptide containing both C and NC possiblemotifs. Our previous work (Miras et al., 2017b) identi-fied Cm eIF4E residues implicated in binding to the C

domain of Cm eIF4G. Amino acid replacements in thecorresponding positions resulted in reduction of theCmeIF4E affinity for Cm eIF4G980–1159 and, concomitantly,reduced cap-independent translation activity of a reportermRNA carrying the viral Ma5TE cap-independenttranslation enhancer (CITE; Miras et al., 2017b). Thestructural analysis shown here indicates that Cm eIF4G,similarly to metazoan eIF4G, binds eIF4E through the Cbut also through the NC domain. Our in vitro andin vivo functional analyses support the importance ofboth motifs for a stable eIF4E-eIF4G complex forma-tion. As in the case of metazoan eIF4E-eIF4G inter-action, our findings may have important practicalimplications, because the plant eIF4E-eIF4G complexis involved in a significant number of plant diseasescaused by viruses (Truniger and Aranda, 2009;Sanfaçon, 2015). In light of our results, a universaleukaryotic bipartite mode of binding to eIF4E isproposed.

RESULTS

Crystal Structure of Cm eIF4E Bound to eIF4G1003-1092

To gain insight into the binding mode of plant eIF4Gto eIF4E, we crystallized and solved the structures ofCm eIF4E in its free form, in the m7GDP cap analogbound state, and in complexwith aCm eIF4Gpeptide to2.2 Å, 3.5 Å, and 1.9 Å resolution, respectively. Previousstructural studies with other eIF4Es from diverse or-ganisms have shown that the full-length protein is re-sistant to crystallization. However, N-terminallytruncated versions of eIF4E, retaining their cap-bindingabilities, have been successfully crystallized (Marcotrigianoet al., 1997, 1999;Matsuo et al., 1997;Monzingo et al., 2007).We expressed an N-terminally truncated Cm eIF4E forcrystallization starting at amino acid 51, thus lacking theputative N-terminal flexible region (eIF4E51-235). The CmeIF4G peptide used for complex formation and crystal-lization, Cm eIF4G1003-1092, contained the canonical eIF4Ebinding motif and a downstream extra sequence thatincluded a hydrophobic patch conserved in other eIF4G,eIFiso4G, and m4E-BP proteins (Fig. 1; SupplementalFig. S1).

The solved structures of Cm eIF4E (Fig. 2), in theirfree state and in complex with a cap analog m7GDP, aresimilar to those observed for other eIF4Es from diverseorganisms (Marcotrigiano et al., 1997; Gross et al., 2003;Monzingo et al., 2007; Paku et al., 2012; Papadopouloset al., 2014; Peter et al., 2015a; 2015b). The Cm eIF4Ecrystals contain four independent copies in the asym-metric unit, all adopting a crescent-shaped conforma-tion formed by a strongly bent beta sheet composed ofeight antiparallel b-strands. The convex surface is dec-orated by three a-helices that form the dorsal surface(Fig. 2A). Major differences are confined to the cap-binding pocket, located on the concave, ventral sur-face of eIF4E. In all the structures described so far, thispocket is built by two conserved Trp residues located in

Plant Physiol. Vol. 174, 2017 1477

eIF4E Structure in Complex with an eIF4G Peptide

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

loops L1 and L3, respectively. In Cm eIF4E and CmeIF4E-m7GDP structures only loop L3 is visible (Fig.2A) and adopts a conformation that closely resemblesthose described for other eIF4E structures in complexwith a cap analog (Marcotrigiano et al., 1997, 1999;Matsuo et al., 1997; Monzingo et al., 2007). The orien-tation adopted by residue W128 is fully compatiblewith cap binding in all structures, though, in ourstructure, the m7GDP cap analog is observed in onlytwo of the four independent molecules in the asym-metric unit interacting with W128 located in loop L3(Fig. 2A). Binding is additionally stabilized throughinteractions between the phosphates in the cap andcharged residues R114, R178, and K183. In contrast,loop L1was never observed in Cm eIF4E and Cm eIF4E-m7GDP structures except for molecule C, in which itadopts a fixed conformation positioning the loop faraway from the binding pocket due to intermolecularcontacts with a neighboring subunit within the asym-metric unit. When Cm eIF4E is bound to the Cm eIF4Gpeptide (Fig. 2, B–G), the formation of a disulfide bondbetween Cys residues 133 and 171 is observed. Thisintroduces a structural rearrangement in loop L3, whichpromotes the formation of a helix 310 (from residue P131to residue A134) and pulls W128 out of the cap-bindingcavity (Fig. 2D), thus rendering a putatively “auto-inhibited” conformation, unable to bind the cap. Eventhough the two Sϒ atoms are also in proximity (4.1 Å) inthe free Cm eIF4E structure, no disulfide bridge wasobserved, and any attempts to crystallize a Cm eIF4Fcomplex without the disulfide bond failed.

EIF4G Exploits Both the C and NC Domains for Bindingto eIF4E

The initial differencemaps of theCm eIF4E-eIF4G1003-1092complex clearly showed the presence of an extracontinuous electron density beyond the C domainextending to the lateral hydrophobic surface of CmeIF4E (Supplemental Fig. S2). Taking thewell-characterizedC domain as an anchoring point, we were able to fit37 amino acid residues of Cm eIF4G (from K1057 toN1093) within this density (Fig. 2, B and C). Thestructure revealed an N-terminal a-helix and a loopconnected by a linker region (Fig. 2, E–G). Each of thesethree structural elements binds to a defined Cm eIF4Esurface: (1) The N-terminal a-helix is formed by the Cdomain and interacts with the dorsal surface of CmeIF4E (Fig. 2E); (2) an elbow after the canonical a-helixthat bends the peptide backbone by approximately 90°,orienting the linker region downward to engage thelateral surface of Cm eIF4E (Fig. 2F); and (3) the loopcontacts a hydrophobic pocket on the lateral surface ofCm eIF4E, indicating the existence of a NC bindingmotif (Fig. 2, E–G). Importantly, this second motif hasbeen identified in m4E-BPs from D. melanogaster and invertebrates (Kinkelin et al., 2012; Peter et al., 2015a,2015b), and very recently in metazoan eIF4G (Grüneret al., 2016). This binding mode is structurally analo-gous to that observed in metazoan eIF4G and m4E-BPs(Supplemental Fig. S3) and is clearly distinct fromthe bracelet-like structure formed in the Sc eIF4E-eIF4G393-490 complex (Supplemental Fig. S3C).

As expected, the C motif of Cm eIF4G presents ana-helical fold that extends from residue Y1059 to A1068(Fig. 2E), which resembles all the canonical peptides incomplex with eIF4E solved so far (Gross et al., 2003;

Figure 1. Structure-based sequence alignment of the eIF4E-interacting regions of eIF4G and m4E-BP orthologs. A sequencealignment from superposition of the different eIF4E crystal structures in complex with eIF4E interacting proteins from highereukaryotes available at the Protein Data Bank (PDB IDs: Cm eIF4G, this work; Hs eIF4G, 5T46; Dm eIF4G, 5T47; Hs 4E-BP1,4UED; Dm 4E-T, 4UE9; Dm Thor, 4UE8; Dm Mxt, 5ABU) was performed using Chimera (Pettersen et al., 2004). The canonicaland NC eIF4E binding motifs (4E-BM) and the elbow loop are framed. The invariant Tyr residue of the canonical motif is typed inbold red and hydrophobic residues conserved in the NC motif are shadowed in blue. Red open circles above the alignmentindicate key eIF4G residues within the domains interacting with eIF4E and mutated in this study. The conserved Ser residue inmetazoan 4E-BPs is shown in bold blue. The two conserved Ser and Thr residues present in metazoan 4E-BPs implicated inphospho-regulation are colored in orange. Sequences are from Cucumis melo (Cm), Homo sapiens (Hs), and Drosophilamelanogaster (Dm).

1478 Plant Physiol. Vol. 174, 2017

Miras et al.

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Mizuno et al., 2008; Umenaga et al., 2011; Kinkelin et al.,2012; Paku et al., 2012). Thus, the interactions establishedbetween the a-helical region containing the consensussequence Y(X)4Lf and eIF4E are highly conserved; thehydroxyl group of the side chain of Y10594G is hydrogen-bonded to the carboxyl oxygen of P614E within the con-served H60-P61-L62 motif in eIF4E and additionallyestablishes lateral hydrophobic interactions with

F10634G. L10644G is buried in a hydrophobic pocketbuilt by Cm eIF4E residues V95, W99, L157, and I160.The next residue defined by the consensus sequenceL10654G makes contacts with W994E and L1574E. In ad-dition, the side chain of residue R10614G contacts Y1544E,while F10674G contacts the lateral chain of H604E andW994E (Fig. 2E). All these residues are well conservedin all eukaryotic eIF4Es and participate in other

Figure 2. Structure and molecular detailsof Cm eIF4E unbound or bound to CmeIF4G1003-1092. A, Ribbon diagram of CmeIF4E51-235 in complex with the m7GDPcap analog. The m7GDP is located in thecap-binding pocket. ResidueW128, in directinteraction with the cap, is marked. B andC, Structure of the Cm eIF4E-eIF4G1003-1092

complex shown as a cartoon and surfacerendering, respectively. Cm eIF4G1003-1092

(orange) contains the C and NC eIF4E bind-ing motifs (4E-BM). D, Structural superposi-tion ofCm eIF4E (in gray as cartoon) andCmeIF4E-eIF4G1003-1092 (in blue as cartoon)binding pocket structures. The formation ofa disulfide bridge (shown as sticks in yellowin the structure) promotes the formation of ahelix 310 in loop L3 and pulls W128 fromm7GDP-interacting position 1 to an appar-ently “auto-inhibited” position 2 (both la-beled in the figure). E, Close-up views of theinteraction of theCm eIF4G Cmotif (orange)with the dorsal surface of Cm eIF4E (blue) intwo orientations. F, Close-up view of the CmeIF4G linker region (orange) and interactionswithCm eIF4E (blue).G, Close-up viewof theNC interaction loop of Cm eIF4G (orange)showing all interactions with the lateral sur-face of Cm eIF4E (blue). Selected interfaceresidues are shown as blue sticks for CmeIF4E and as orange sticks forCm eIF4G. InE to G, residues implicated in the interactionsbetweenCm eIF4E andCm eIF4G1003-1092 areshown as sticks.

Plant Physiol. Vol. 174, 2017 1479

eIF4E Structure in Complex with an eIF4G Peptide

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

eIF4Gs and m4E-BPs interactions through the C motif(Supplemental Fig. S4;Marcotrigiano et al., 1999; Kinkelinet al., 2012; Peter et al., 2015a, 2015b).

The characteristic elbow loop, present in m4E-BPsand metazoan eIF4Gs, which begins immediately afterthe R/Q residues at position 9 of the canonical motifs(HsQ621 andDm R630) and contains a half helical turnbeginning at the tip of the elbow (Hs F624-S626 andDmK633-S635), is not observed in our structure. Instead,the a-helix containing the C domain presents an extraturn-reaching residue L10724G, which is stabilized throughmain-chain interactions between residues A10684G andF10714G and the side chain of residue N1034E (Fig. 2F).Hydrophobic interactions are also established betweenthe side chains of residues A10684G and F10714G andresidues W994E and E964E. This extra helical turnslightly bends the helix toward the lateral surface of CmeIF4E and thus allows the NC domain of Cm eIF4G tocontact the hydrophobic lateral core of Cm eIF4E (Figs.2G and 3A). An intramolecular network of hydropho-bic interactions is built by Cm eIF4G residues L1074,F1078, V1080, I1084, and M1088. I894E, a conservedhydrophobic residue in eukarya, is central in the in-teraction with Cm eIF4G and provides a hydrophobiccore around which hydrophobic residues F10784G andI10844G are arranged (Fig. 3A). On the beta-turn ofCm eIF4G, L1087 contacts the main-chain conserved

residues F704E and I1054E and engages the hydrophobicpocket also with L1114E and L1174E. M10884G makesside chain contacts with I1054E and with the carbonyloxygen of H1064E. In the N terminus of the NC motif,F10784G contacts V1014E of a-helix a1 and Y904E ofb-strand b1. In addition to the hydrophobic interac-tions, S10894G and T10924G show hydrogen bonds withN1044E located on helix a1 (Fig. 2G). The last C-terminalextension is stabilizedmainly through hydrogen bonds.

Despite the apparent lack of sequence conservation inthe NC motif of eIF4Gs and m4E-BPs, the structure-based amino acid sequence alignment indicates thatthis patch of hydrophobic residues within this do-main is mostly conserved between plants, verte-brates, and insects (Fig. 1; Supplemental Fig. S1). Infact, the spatial arrangement of residues F10784G,I10844G, and L10874G in our structure closely relatesto those observed in H. sapiens and D. melanogastercomplexes (Fig. 3). More specifically, residues L6334G,I6364G and V6394G and V6414G, I6464G and L6474G inthe human and D. melanogaster eIF4F complexes occupyequivalent positions (Fig. 3, B and C; Grüner et al., 2016).This cluster of hydrophobic interactions is also observed,although to a lesser extent, in otherm4E-BPs (Fig. 3,D–H);the highly conserved eIF4E hydrophobic pocket inter-acts with side chains of Thor V75 and L79, 4BP1 V81,4E-T F41 and W42, Mxt M605, and I373 or Cup M371

Figure 3. Structures of the eIF4E lateral hydrophobic pocket interacting with eIF4G and m4E-BP peptides. Hydrophobicity ofresidues in all eIF4E-interacting domains is conserved. Close-up views of eIF4E hydrophobic patch and residues involved in thebinding of Cm eIF4E and eIF4G1003-1092 (A), Hs eIF4E and eIF4G592-653 (B; PDB ID: 5T46; Gruner et al., 2016), Dm eIF4E andeIF4G601-660 (C; PDB ID: 5T47; Gruner et al., 2016), Dm eIF4E and m4E-BP Thor50-83 (D; PDB ID: 4UE8; Peter et al., 2015a), HseIF4E and m4E-BP 4E-BP150-83 (E; PBD ID: 4UED; Peter et al., 2015a), Dm eIF4E and m4E-BP 4E-T9-44 (F; PBD ID: 4UE9; Peteret al., 2015a), Dm eIF4E and m4E-BP Mxt577-640 (G; PBD ID: 5ABV; Peter et al, 2015b), and Dm eIF4E and m4E-BP Cup325-376(H; PBD ID: 4AXG; Kinkelin et al., 2012).

1480 Plant Physiol. Vol. 174, 2017

Miras et al.

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

and I373 (Fig. 3, D–H; Peter et al., 2015a, 2015b). Thus,binding to the lateral hydrophobic surface of eIF4Eappears to be a conserved feature of m4E-BPs, eukar-yotic eIF4Gs, and plant-specific eIFiso4G isoforms.

Functional Validation of the Cm eIF4E-eIF4G1003-1092Structure

To validate our structural results, we substituted keyresidues in both proteins and performed in vitro pull-down assays (Fig. 4). We tested recombinant frag-ments of wild-type Cm eIF4G1003-1092 (eIF4G-WT) andmutated versions of it for binding to full-length CmeIF4E. Mutations were introduced in the C (eIF4G-C),the NC (eIF4G-NC), and both (eIF4G-C-NC) Cm eIF4E-binding motifs. In the well-conserved C motif Y(X)4Lfof eIF4G-C, three residues, Y1059, L1064, and L1065,were substituted by Ala, while the hydrophobic resi-dues F1078, I1084, and L1087 of the proposed NCmotifin eIF4G-NC were substituted by Asp residues. AlleIF4G fragments were expressed in Escherichia colias N-terminal fusions of the maltose binding protein(MBP), and purified Cm eIF4E was added to the CmeIF4G1003-1092 (wild type and mutants) cleared lysates ina fixed concentration. This purely qualitative pull-down analysis showed that substitutions in either theC or the NC binding-motifs did not disrupt the associ-ation of Cm eIF4G1003-1092 with Cm eIF4E (Fig. 4, lanes3–8), while no binding to Cm eIF4E was observed whenamino acid substitutions in the C and NC domains(eIF4G-C-NC) were combined (Fig. 4, lanes 9–10). Toconfirm these results, we compared the binding effi-ciencies of each mutant in pull-down assays using in-creasing concentrations (0.5–3 mM) of Cm eIF4E witha fixed concentration of Cm eIF4G1003-1092 (Fig. 5). Inagreement with the results above, EIF4G-WT andeIF4G-NC seemed to display similar affinities for Cm

eIF4E, binding Cm eIF4E at the lowest concentration(Fig. 5, A and D; MBP control in Fig. 5B), while theeIF4G-C interaction with Cm eIF4E was clearly im-paired (Fig. 5C). Again, no binding was observed forthe eIF4G-C-NCmutant, even ifCm eIF4Ewas added inexcess (Fig. 5E). We quantified band intensities of thepulled-down Cm eIF4E and plotted them versus the CmeIF4E input (Fig. 5F). When a hyperbolic binding curvewas fitted to the resulting concentration data, approx-imate dissociation constants (Kd) of 2.15, 2.67, and1.90 mM were obtained for eIF4G-WT, eIF4G-C, andeIF4G-NC, respectively. Therefore, in our assay, each ofthe binding sites seemed to be sufficient for a similarlyproductive interaction, and only simultaneous muta-tions affecting the two binding sites resulted in bindingdisruption. It should be pointed out that there are im-portant differences in the Kd values estimated here andthe value estimated for the wheat (Triticum aestivum)full-length eIF4E-eIF4G complex (Mayberry et al.,2011), and these may result from the nature of the CmeIF4G peptide versus thewheat full-length protein; still,our data are fully useful for the relative comparison ofthe Cm eIF4G peptides analyzed here.

To further validate these results, we examined theeffect of substitutions in Cm eIF4E residues proposed tobe involved in Cm eIF4G binding, including F70A,I89A, W99A, and Y154H, even as double and triplemutants Y154H-W99A, F70A-I89A, Y154H-W99A-F70A, and Y154H-I89A-F70A. Lysates of MBP-taggedeIF4G-WT, and wild-type or mutant Cm eIF4Es wereincubated and pulled-down using the MBP-tag. In thiscase,Cm eIF4Ewas visualized bywestern-blot analysis.Substitutions W99A and Y154H-W99A on the dorsalsurface of Cm eIF4E reduced its Cm eIF4G1003-1092-binding capacity to 70% and 33%, respectively, com-pared to eIF4E-WT (Fig. 6, lanes 3 and 5). On the otherhand, the substitution of the hydrophobic residues ofthe lateral pocket of Cm eIF4E, F70A, and double mu-tant F70A-I89A reduced its Cm eIF4G1003-1092-bindingefficiency to 77% and 43% compared to eIF4E-WT, re-spectively (Fig. 6, lanes 6 and 8). Interestingly, bindingwas affected to similar extents for single and doublemutants in dorsal and lateral surfaces. When substitu-tions in both surfaces were combined, binding wasstrongly impaired, resulting in 30% and 21% of theeIF4E-WT efficiency (Fig. 6, lanes 7 and 9), respectively.These results suggest that Cm eIF4G needs to interactwith both the dorsal and lateral surfaces ofCm eIF4E forefficient binding.

Testing Substitutions in Cm eIF4E-Binding Residues in aTranslation Efficiency Assay

Recently, we developed a translation efficiency assayto test Cm eIF4E mutants in vivo (Miras et al., 2017b).This assay is based on the RNA element called Ma5TElocated in the 39-UTR of Melon necrotic spot virus(MNSV, family Tombusviridae), which requires and hasa strong specificity for Cm eIF4E alleles to efficiently

Figure 4. In vitro interaction of Cm eIF4E and Cm eIF4G1003-1092. MBPpull-down assay showing the interaction of Cm eIF4E (wild type, fulllength) and MBP-Cm eIF4G1003-1092 (either wild type or mutated in C,NC, or C-NC). Bacterial lysates expressingMBP-taggedCm eIF4G1003-1092

(wild type and mutants) were incubated with purified Cm eIF4E 1 mM in avolume of 300mL.Of these, 15mL (5%; input) was analyzed by SDS-PAGEfollowed by Coomassie Brilliant Blue staining. Bound proteinswere elutedin 60 mL, and 10 mL (16%; pulldown) was loaded on the gel. MBP servedas a negative control (lanes 1 and 2).

Plant Physiol. Vol. 174, 2017 1481

eIF4E Structure in Complex with an eIF4G Peptide

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

drive cap-independent translation of the messengerRNAs bearing it (Nieto et al., 2006; Truniger et al., 2008;Rodríguez-Hernández et al., 2012). Briefly, this assay isbased on the capacity of Cm eIF4E-WT to complementthe Ma5TE-controlled translation of RNAs when itis transiently expressed in melons that carry the CmeIF4EH228L allele in homozygosity, which does notsupport Ma5TE-driven translation (Nieto et al., 2006;Miras et al., 2017b). Thus, we transiently expressed CmeIF4E mutants in eIF4EH228L melons and studied their

ability to complement Ma5TE-controlled translation ofreporter RNAs consisting of the luciferase gene flankedby the 59-and 39-UTRs of MNSV.

Recapitulating previous results (Miras et al., 2017b),our data showed that transient expression of CmeIF4E-WT increased Ma5TE-driven translation 6-foldwith respect to translation in its absence (Fig. 7, (-) andwild type), reaching nearly 50% of the translation ac-tivity of an eIF4E-independent reporter RNA used tonormalize the data (see “Materials and Methods” for

Figure 5. Binding affinity ofCm eIF4E andCm eIF4G1003-1092.MBP pull-down assays showing the interaction of purifiedCm eIF4E(wild type, full length; increasing concentrations, 0.5–3 mM) and MBP-tagged Cm eIF4G1003-1092 (wild type or mutant in bacteriallysates at a fixed concentration). A, Pull-downwithCm eIF4G1003-1092-WT. B, Pull-downwithMBP used as a negative control. C toE, Pull-down with MBP-eIF4G1003-1092 mutants in C, NC, and C-NC domains. EIF4G lysates (wild type and mutants) were in-cubated with purified Cm eIF4E at concentrations of 0.5, 1, 2, and 3 mM (corresponding to lanes 1, 2, 3, and 4 of each gel, re-spectively) in a volume of 300 mL. The input samples (lanes 1 to 4) and pull-down fractions (lanes 5 to 8) were analyzed in a 12%SDS-PAGE followed by Coomassie Brilliant Blue staining, loading in gels 5% and 16% of the total volumes, respectively. F, Graphrepresenting the specific binding of Cm eIF4E with Cm eIF4G1003-1092 (wild type and mutants), plotted as a function of Cm eIF4Econcentration. Data points in the binding curves represent means calculated from data points of two different experiments as in A,C, and D. Each data was fitted using a one-site binding model.

1482 Plant Physiol. Vol. 174, 2017

Miras et al.

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

details); in contrast, transient overexpression of CmeIF4EH228L did not support translation (Fig. 7, H228L).We then studied the effect on translation of the substi-tutions that reduced Cm eIF4G-binding in vitro. Singleand double substitutions in residues of the dorsal sur-face of Cm eIF4E, in contact with the C domain of CmeIF4G, resulted in a drastic reduction of the Ma5TE-driven translation efficiency (Fig. 7, W99A and Y154H-W99A; Miras et al., 2017b). In contrast to in vitro bindingassays, substitutions in the lateral surface of Cm eIF4E,involved in interactionswith the NC binding domain inCm eIF4G, also reduced translation efficiency (Fig. 7,F70A and F70A-I89A). Similarly, Cm eIF4E-mutantswith substitutions in residues at both surfaces wereunable to complement translation in this assay (Fig. 7,Y154H-F70A-I89A and Y154H-W99A-F70A). These re-sults strongly support the importance of the Cm eIF4E

residues implicated in binding to Cm eIF4G through theNC domain for eIF4F-dependent translation. The in-teraction of both Cm eIF4E surfaces with Cm eIF4G isrequired for efficient translation.

For the mutant proteins tested here, we studied theirability to complement the growth of the eIF4E-deficientyeast strain JO55. This Saccharomyces cerevisiae strainlacks its endogenous eIF4E gene and requires comple-mentation with an external eIF4E to survive, for examplethe human eIF4E expressed from the pGAL-eIF4E-URA3plasmid in a Gal-dependent manner (Altmann et al.,1989). This assay is a good indicator of all the eIF4Efunctions that affect yeast growth, including mRNAtranslation (Altmann et al., 1989; Charron et al., 2008;German-Retana et al., 2008; Ashby et al., 2011), and wehave used it previously to test the effect of single CmeIF4E mutations (Miras et al., 2017b). Almost all Cm

Figure 6. Recovery of Cm eIF4E (wild type and mutants) in pull-down assays with MBP-Cm eIF4G1003-1092. Pull-down assaysshowing the interaction of wild-type and mutant Cm eIF4E with MBP-Cm eIF4G-WT. A fixed volume of a lysate from bacteriaexpressing Cm eIF4G1003-1092-WT with a N-terminal MBP tag was incubated with Cm eIF4E bacterial lysates (wild type andmutants) in a volume of 300mL.Middle, Pulled-downwild-type andmutantCm eIF4Es as determined byWestern blot (WB) usingaCm eIF4E-specific antibody. Top, The same gelwas stainedwith Coomassie Brilliant Blue to compare the amount of elutedMBP-tagged Cm eIF4G-WT in each experiment (Coomassie). Bottom, The amount of Cm eIF4E present in the input visualized bywestern blot. Bands from WB were quantified and a binding was estimated using the band intensity ratio between sample CmeIF4E-WT (input, lane 5) and Cm eIF4E-WT (MBP pull-down, lane 4) as 100% reference value. The interacting factors in eachexperiment are shown below the gels: pull-down proteins, eitherMBPorMBP-Cm eIF4G, and interactingCm eIF4E proteins (wildtype or mutants). Input and pull-down correspond to a 1% of the incubation volume and 5% of the elution volumes, respectively.

Plant Physiol. Vol. 174, 2017 1483

eIF4E Structure in Complex with an eIF4G Peptide

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

eIF4E mutants analyzed here complemented yeastgrowth, confirming their functionality in this organism(Supplemental Fig. S6), with the exception of the triplemutant Y154H-F70A-I89A (Supplemental Fig. S6) har-boring substitutions in two residues involved in NCand one residue in canonical binding to Cm eIF4G.

DISCUSSION

Architecture of the Cap Binding Site

All the structural information available to date showsthat the eIF4E binding pocket is built by two principalregions when bound to either the m7GDP or m7GTPcapanalogs; a conservedTrppair located in the loop regionand a cluster of Arg/Lys residues interacting with thephosphates in the cap. NMR cap-free eIF4E structuresshow that loops L1 and L3 are quite flexible and adoptmultiple conformations in solution (Volpon et al., 2006).However, they get ordered upon binding the capthrough stacking interactions with these two Trp resi-dues each located in each loop (Marcotrigiano et al.,1997, 1999; Matsuo et al., 1997; Monzingo et al., 2007).As mentioned before, in our structure only loop L3 isvisible and adopts a conformation, either in its m7GDP

bound or unbound state, that closely resembles thosedescribed for other eIF4E structures in complex witha cap analog and thus positioning residue W128 in aconformation fully compatible with cap binding. Adefined conformation for both loops has been observedin the crystal structure of the human eIF4E in its un-bound state, with the four eIF4E molecules in theasymmetric unit arranged as dimers and the static po-sition of both loops due to intermolecular contacts be-tween the concave surface of both monomers in eachdimer (Siddiqui et al., 2012). Whether the static con-formation observed for loop L3 in our structure isdriven by such contacts is difficult to infer, since thereare four independent Cm eIF4E molecules in theasymmetric unit that result in a different local envi-ronment for each loop. In contrast, loop L1 was neverobserved in our Cm eIF4E and Cm eIF4E-m7GDPstructures except for molecule C, in which it adopts afixed conformation. However, this appeared to be aconformational selection triggered by crystal packingrather than a functionally relevant state in terms of capbinding, since it positions the loop far away from thebinding pocket. It should be noted that after structurerefinement, local distribution of B factors in this regionindicated that the binding pocket was not fully occu-pied in the two Cm eIF4E molecules in which we

Figure 7. Effect of substitutions in Cm eIF4E on cap-independent translation efficiency. Relative luciferase activity (%) obtainedwith a reporter RNA that depends on Cm eIF4E for cap-independent translation (see inset in the right upper part of figure) melonprotoplasts. Protoplasts carried Cm eIF4EH228L, which is unable to efficiently complement cap-independent translation of thereporter RNA. Protoplasts were transiently transformed to express different Cm eIF4E variants (wild type, which is able tocomplement cap-independent translation of the reporter;Miras et al., 2017b; andmutants). For normalization ofmeasurements indifferent protoplast preparations, luciferase activity obtained with a Cm eIF4E independently translated reporter RNA construct59-UTR-luc-39-UTR was set to 100% for each protoplast preparation. The transiently expressed Cm eIF4E mutants are indicatedbelow each bar: -, silencing suppressor P19 alone; WT, wild type; engineered Cm eIF4E mutations, H228L, W99A and Y154H-W99A (in residues in the dorsal surface proposed to be involved in canonical Cm eIF4G interaction; Miras et al., 2017b), andF70A and F70A-I89A (in residues in the lateral surface proposed to be involved in NC Cm eIF4G interaction); and mutations inboth surfaces Y154H-F70A-I89A and W99A-Y154H-F70A. Error bars are 6SD. Panels below the graph show expression of eachCm eIF4E mutant in melon cotyledons visualized by western blot using antibodies against Cm eIF4E. Bottom, Loading as visu-alized by Coomassie Brilliant Blue staining.

1484 Plant Physiol. Vol. 174, 2017

Miras et al.

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

observed extra density, so occupancy for m7GDP wasfinally adjusted to a value of 0.6 to match local B factordistribution. It is plausible then, that the local concen-tration of m7GDP in the binding pocket is too low todrive the transition of loop L1 to its cap-binding con-formation, while loop L3 is already in a competentconformation to interact with the guanine base of thecap. Structural studies in solution should be performedin the future to analyze the flexibility of these loops inCm eIF4E.

A Universal Bimodal Binding Mode of eIF4E to eIF4G inHigher Eukaryotes?

This work provides the first description of thestructure of a plant eIF4G peptide in complex witheIF4E and shows that it binds to both dorsal and lateralsurfaces of Cm eIF4E interacting through the C and NCdomains, similarly to metazoan eIF4Gs and 4E-BPs(Kinkelin et al., 2012; Peter et al., 2015a, 2015b; Grüneret al., 2016). Using in vitro pull-down assays withproteins mutated in these domains, we showed thatmotifs in both domains were required for efficientcomplex formation. Also, our functional analysis usingan in vivo cap-independent eIF4E-dependent translationsystem (Miras et al., 2017b) supports the importance ofthis bimodal type of binding.The eIF4G residues belonging to the C motif are

generally conserved in eukaryotic eIF4Gs (Miras et al.,2017b). In contrast, the residues of the NC motif iden-tified here are not conserved, but the hydrophobicity of

the lateral eIF4E core and of the NCmotif are conservedin eukaryotic eIF4G andm4E-BPs (Fig. 1; SupplementalFig. S4). Moreover, this patch of hydrophobic residueswithin the NC domain is also conserved in the plant-specific eIFiso4G, suggesting a similar bimodal inter-action in the isoform complex eIFiso4F (SupplementalFig. S1). Thus, our structure of the Cm eIF4E-eIF4G1003-

1092 complex very much resembles those of metazoancomplexes, where the canonical helix is bound to thedorsal surface of eIF4E while binding extends to thelateral interface (Supplemental Fig. S3, A and B; Grüneret al., 2016). Melon and metazoan complexes are struc-turally similar, but differ from that of yeast (SupplementalFig. S3C; Gross et al., 2003; Grüner et al., 2016). This dif-ference could be due to the lack of sequence conservation.However, the yeast eIF4E-eIF4G393-490 structure was de-termined by NMR in the presence of a zwitterionic de-tergent that may interfere with hydrophobic bonds(Matsuo et al., 1997; Gross et al., 2003; Grüner et al., 2016).Therefore, our data together with those of Grüner andcoworkers (Grüner et al., 2016) strongly support a uni-versal bimodal binding mode of eIF4E to eIF4G in highereukaryotes.

Peculiarities of the Plant eIF4E-eIF4G1003-1092 Complex

A search for structural neighbors within the databaseof the Dali server revealed that Drosophila Mextli pro-tein (Dm Mxt) shares its topology with Cm eIF4G at itsmaximum score (Z = 2.4), even higher than Dm and HseIF4G (Z = 1.9 and Z = 1.6, respectively). In plants, the

Figure 8. Structural comparison of CmeIF4G, metazoan eIF4Gs, and 4E-BP linkerregions. A, Extended consensus sequencefor the plant eIF4G canonical eIF4E bindingdomain from all available databank se-quences (Patrick and Browning, 2012) andDm Mxt and consensus m4E-BPs. Invariantresidues are typed in bold, and plant-specificAla or Ser residues in position 9 (boxed) arered colored. B to F, Close-up views of thecanonical helices and the linker region of CmeIF4G (B), Dm Mxt (C), Dm eIF4G (D), HseIF4G (E), andDm Thor (asm4E-BPmodel; F)bound to the dorsal surface of eIF4E (PDB IDs:5T46, 5T47, 5ABV, 4UE8; Peter et al., 2015a,2015b; Gruner et al., 2016). Residues of thecanonical helix in position 9 are shown assticks.

Plant Physiol. Vol. 174, 2017 1485

eIF4E Structure in Complex with an eIF4G Peptide

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

eIF4G C motif can be extended to the consensus se-quence [YSRDFLLX2(A/S)] (Patrick and Browning,2012; Fig. 8A), in which Cm eIF4G position 10 (under-lined) is occupied by an Ala (A1068). Interestingly,while metazoan eIF4Gs and most of the m4E-BPs con-tain long aliphatic residues (K/Q/R) at this position, inDmMxt, shorter residues (Ile or Ser) are present, whichallows for a tripartite binding mode for Dm Mxt (Peteret al., 2015b); an auxiliary helical motif can be set on thedorsal surface of eIF4E, replacing the interactions andcovering the surface occupied by the side chains of thealiphatic residues present in the majority of m4E-BPsand metazoan eIF4Gs (Fig. 8, B–F). Thus, it may behypothesized that plant eIF4Gs may use this tripartitebinding mode, making the complexes more stable andgranting them with distinct functional properties.

As mentioned before, the characteristic half-helicalturn beginning at the tip of the elbow loop (Hs F624-S626and Dm K633-S635) present in all the metazoan 4E-BPsand eIF4Gs structures solved so far was not observed inthe Cm eIF4E-eIF4G1003-1092 complex (Supplemental Fig.S3). The contact established between the side chainsof the highly conserved S/T residues located at theend of the elbow loop in metazoan eIF4Gs and 4E-BPsand carbonyl oxygens of the preceding residues fixingthe backbone were lost in our structure (Peter et al.,2015a). Instead, the side chain of Cm F10714G occupiedthe position of the S/T residue present in its metazoancounterparts, introducing a two-amino acid gap (Figs. 2and 9A), and explaining why the half-helical turn is lostin the Cm eIF4E-eIF4G1003-1092 structure. Interestingly,two conserved S/T residues have been located at bothends of the canonical elbow loop in all the m4E-BPcrystal structures solved so far (Fig. 9, B–D; Peter et al.,

2015a; Grüner et al., 2016). The phosphorylation ofthese linker residues, not present in metazoan eIF4Gs(Fig. 1), is sufficient to reduce the strength of m4E-BPsas translational repressors and the mechanism behindthis appears to be a phosphorylation-induced disorder-to-order transition that drastically reduces the affinityof m4E-BPs for eIF4E (Bah et al., 2015). In plants, onlyLOX2 and BTF3 have been identified as putative eIF4Einteracting proteins (Freire et al., 2000, 2005). WhileBTF3 does not contain an eIF4E-recognizable bindingmotif, LOX2 possesses a sequence that resembles a Cdomain, but it is poorly conserved. In contrast to m4E-BPs, there is no apparent hydrophobic patch that wouldcorrespond to a NC interacting region (SupplementalFig. S5). Moreover, as in the case of Cm eIF4G, the S or Tresidues are not conserved downstream of the C do-main from LOX2. Thus, the lack of a “metazoan-like”elbow loop and the fact that the characteristic phos-phorylable S/T residue pairs in metazoans seems not tobe conserved in plants suggest that a different mecha-nismmay control the assembly and stability of the planteIF4E-eIF4G complex.

Another peculiarity seems to be the formation of adisulfide bridge in the Cm eIF4E-eIF4G1003-1092 complex.As expected, the Cm eIF4E structure has much in com-mon with that of mammalian and other plant eIF4Es(Marcotrigiano et al., 1999; Monzingo et al., 2007; Ashbyet al., 2011). Interestingly, themain difference betweenCmeIF4E either in its free or cap-bound state with Cm eIF4Ebound toCm eIF4G peptide is the formation of a disulfidebridge in the Cm eIF4E-eIF4G1003-1092 complex, whichseems to block the ability of Cm eIF4E to bind to the cap(Fig. 2D). The formation of a disulfide bridge has beenpreviously observed in the wheat eIF4E crystal structure,

Figure 9. Structural comparison of the el-bow loops ofCm eIF4G,metazoan eIF4Gs,and 4E-BP. A to D, Close-up views of thecanonical helices and elbow loops of CmeIF4G (A), Hs eIF4G (B), Dm eIF4G (C),and Dm Thor (as a m4E-BP model; D)bound to the dorsal surface of eIF4E (PDBIDs: 5T46, 5T47, 4UE8; Peter et al., 2015a;Gruner et al., 2016). Selected residuesmediating phosphorylation and their ana-logs are shown as sticks.

1486 Plant Physiol. Vol. 174, 2017

Miras et al.

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

where cocrystallization with the cap was only possibleusing an eIF4Emutant inwhich one of the C residueswasreplaced to avoid disulfide bridge formation (Monzingoet al., 2007). Despite that the two cysteines involved arestrictly conserved in plant eIF4Es, suggesting that theymayhave some important biological role, affinity-bindingassays under both reducing and oxidizing conditionsshowed that in both caseswheat eIF4E retained the abilityto bind the cap (Monzingo et al., 2007). However, furtherstudies using mass spectrometry and a Lys-specificchemical probe indicated structural changes upon al-tering the redox state, supporting the hypothesis of aredox sensor or switch for eIF4E (O’Brien et al., 2013).Whether the oxidation state of eIF4Emaymodulate capbinding in a redox-sensing manner in plants and reg-ulate the level of translation in response to the redoxstate of the cell remains uncertain.

Implications in Plant Disease: EIF4G and Potyviral VPgPossibly Share an eIF4E-Binding Surface

Plants harboring mutations in eIF4E lose their sus-ceptibility to a number of plant viruses; thus specificeIF4E alleles confer recessive resistance to viruses(Truniger and Aranda, 2009; Sanfaçon, 2015; Dinkovaet al., 2016). One example is the Cm eIF4E-mediated

resistance of melon to MNSV (Tombusviridae) that re-sults from a single amino acid substitution in this pro-tein (Nieto et al., 2006). The resulting Cm eIF4E variantis still functional for translation of plant mRNAs, but itdoes not promote translation ofMNSVRNAs (Trunigeret al., 2008). We show here that mutations in Cm eIF4Eaffecting its interaction with Cm eIF4G reduce theeIF4E-dependent translation of MNSV RNAs, con-firming that the Cm eIF4E-eIF4G complex is requiredfor MNSV translation (Miras et al., 2017b). However,we cannot rule out that thesemutations could reduce itsability to bind the 39-CITE, even though the Cm eIF4Eresidues mutated here are not in the vicinity of theresidue H228 involved in 39-CITE binding (Miras et al.,2017b). But nearly all of the eIF4E-dependent virusesbelong to the family Potyviridae (Sanfaçon, 2015). Inmany of these cases, the resistance phenotype arisesfrom disruption of the direct interaction between thepotyviral genome-linked protein (VPg) and eIF4E(Léonard et al., 2004; Yeam et al., 2007; Charron et al.,2008; Miras et al., 2017a). Overall, two regions of eIF4Ehave been mapped to be involved in the interactionwith VPg, one near the cap-binding pocket and an-other in the lateral surface of the protein. This lattersurface interacts with the eIF4G NC motif (Fig. 10A),but a C motif could not be identified in VPgs, whichon the other hand are recalcitrant to crystallization

Figure 10. Plant virus resistance mutations modeled on the melon eIF4E-eIF4G1003-1092 complex. A, The residues implicated inplant potyvirus and bymovirus resistance are shaded in green and magenta, respectively (Gao et al., 2004; Kang et al., 2005;Kanyuka et al., 2005; Ruffel et al., 2005; Stein et al., 2005; Nicaise et al., 2007; German-Retana et al., 2008; Ashby et al., 2011).The Cm eIF4Emolecule is shown as a surface rendering and Cm eIF4G1003-1092 is shown as a cartoon representation, both coloredin gray and orange, respectively. Them7GDP cap-analog is located in the cap-binding pocket ofCm eIF4E. B, Sequence alignmentof Cm eIF4G (residues 1052–1093) and potyviral VPgs (ZYMV NP477522.1; TEV NP734204.1; LMV KF285932.1; TuMVNP734219.1; PRSV AEC04846.1). C and NC motifs are boxed in the Cm eIF4G sequence. Conserved residues are typed in boldand hydrophobic residues located in the putative NC motif are green-colored.

Plant Physiol. Vol. 174, 2017 1487

eIF4E Structure in Complex with an eIF4G Peptide

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

(Rantalainen et al., 2008). Despite the lack of sequenceconservation, an alignment of Cm eIF4G and VPgsidentified three conserved amino acids in a hydropho-bic region that may correspond to a putative NC motif(Fig. 10B). Therefore, eIF4E andVPgsmay bind throughNC motifs. At least one amino acid substitution in thelateral surface of tomato eIF4E, G107R, disrupted inplanta its interaction with VPg from Tobacco etch virus(Yeam et al., 2007). Moreover, analysis of mutations inthe lateral surface of pea (Pisum sativum) and lettuce(Lactuca sativa) eIF4Es revealed that the mutant proteinscould not complement viral infection of two potyvi-ruses, Pea seed-borne mosaic virus and Lettuce mosaic virus(German-Retana et al., 2008; Ashby et al., 2011). Inter-estingly, several studies have suggested that potyviralVPg is able to inhibit cap-dependent translation (Khanet al., 2008; Miyoshi et al., 2008; Eskelin et al., 2011).Thus, it can be hypothesized that VPg competes witheIF4G for binding to the lateral surface of eIF4E throughits NC binding motif, impairing eIF4E-eIF4G interac-tion and hijacking eIF4E for viral genome translation.However, it should be pointed out that apart fromtranslation, other roles have been proposed for the VPg-eIF4E interaction, including viral cell-to-cell movement(Gao et al., 2004).

In conclusion, we have identified and characterizedkey structural principles determining plant eIF4E-eIF4G interaction. The identification of extended andconserved interactingmotifs supports a universal bindingmode in eukaryotes and offers targets for the design andedition of eIF4E genes to regulate translation and blockviral infection.

MATERIALS AND METHODS

Plasmids

The plasmids expressing Cm eIF4G-WT and Cm eIF4E were previouslydescribed (Miras et al., 2017b). Briefly, Cm eIF4G (residues 1003–1092) and CmeIF4E (residues 51–235) were amplified by PCR from plasmids pTOPO-Cm-4Gand pET15b-4EVed (Nieto et al., 2006) and cloned by LIC technology into thep2CT (which provides an N-terminal MPB tag followed by a TEV proteasecleavage site) and p2AT expression vectors, respectively (Macrolab, UCBerkeley),yielding p2CT-eIF4G-p10 and p2AT-eIF4E-WT (Miras et al., 2017b). For ex-pression of the Cm eIF4E-eIF4G1003-1092 complex, a dicistronic expression vectorwas generated by cloning into plasmid p2D (Macrolab), yielding p2D-4Fp10.Cm eIF4E was cloned into cassette 1 using BamHI/XbaI restriction sites, and CmeIF4G1003-1092-WTwas cloned into cassette 3 using SbfI/AscI restriction sites. Allthe Cm eIF4E and Cm eIF4G mutants were generated by site-directed muta-genesis using plasmids pET15b-4EVed (H228) and p2CT-eIF4G-p10, respec-tively, and the oligonucleotide primers shown in Supplemental Table S1. Fortransient Cm eIF4E expression in the complementation experiments, the CmeIF4E constructs were cloned into the binary vector pBIN61 using XbaI/XmaIrestriction sites. All mutants were confirmed by DNA sequencing. Luc con-struct with the 5 - and 3 -UTR from MNSV-Ma5 and MNSV-264 flanking thefirefly luciferase gene has been previously described (Truniger et al., 2008).Reporter RNA was obtained by in vitro transcription (RiboMAX Large ScaleRNA production, Promega).

Protein Expression and Purification

All proteins were expressed in Escherichia coli Rosetta (DE3) pLysS cells(Novagen) that were grown in Luria-Bertani medium at 37°C to an OD600 of 0.6.At this point, isopropylthio-b-galactoside was added to a final concentration of

0.4 mM to induce protein expression O/N at 20°C. Cells were resuspended innickel buffer A (25 mM HEPES, pH 7.5, 400 mM NaCl, 20 mM imidazole, 10%glycerol, 1 mM dithiothreitol) and supplemented with DNaseI (Roche) andprotease inhibitor cocktail (Roche) and lysed by sonication. Expressed CmeIF4E-eIF4G1003-1092 complex was eluted from a HisTrap HP column (GEHealthcare) using nickel buffer B (same as nickel buffer A but with 400 mMimidazole). After buffer exchange, the eluted protein was quantified by spec-trometry at OD280. The His6 and MBP tags were removed after cleavage withTEV protease at a 1:20 mass ratio and overnight incubation at 4°C. Finally, theprotein was loaded onto the same HisTrap HP column and eluted with Nickelbuffer B followed by loading onto a size-exclusion Superdex 200 16/60 column(GE Healthcare). Cm eIF4E51-235 was expressed from p2A-eIF4E-WT as de-scribed above, purified using a HisTrap HP column (GE Healthcare), andpassed through a final size exclusion chromatography column (Superdex200 16/60, GE Healthcare). Purification of full-length Cm eIF4E was carried outas previously described (Nieto et al., 2006). All proteins were concentrated to afinal concentration of 10mg/mL, and buffers were exchanged to the final buffer25 mM HEPES, pH 7.5, 200 mM NaCl, and 10% glycerol.

Crystallization, Data Collection, and Processing

Crystals were obtained using the hanging drop vapor diffusion method.Briefly, 1 mL of protein solution (10 mg/mL) was mixed with an equal volumeof reservoir solution and incubated at 20°C until crystals suitable for x-raydiffraction were obtained. Cm eIF4E crystals were grown using 1.0 M sodium/potassium tartrate in 100 mM MES, pH 6.0, as the precipitant, and Cm eIF4E-m7

GDP complex was obtained by incubating native crystals O/N in the presenceof 1:1.1 excess ligand. Cm eIF4E-eIF4G1003-1092 complex was crystallized using1.4 to 1.6 M ammonium sulfate, 100 mM sodium acetate, pH 4.5, as the reservoirsolution. Small needles were obtained when plates were incubated at 20°C.Suitable crystals for x-ray data collection were obtained using microseedingtechniques. Data were collected using synchrotron radiation at ALBA-CELLSafter flash freezing under liquid nitrogen using 20% glycerol as the cryo-protectant. Diffraction images from one single crystal for each complex were pro-cessed using XDS (Emsley and Cowtan, 2004) and SCALA (Collaborative, 1994).

Cm eIF4E-eIF4G1003-1092 Structure Solution and Refinement

Initial phaseswere determined bymolecular replacementwith theMOLREPprogram (Collaborative, 1994) using the crystal structure of wheat (Triticumaestivum) eIF4E as a search model (Monzingo et al., 2007; PDB ID: 2IDR). Rigidbody refinement was conducted using REFMAC5 (Murshudov et al., 1997).Manual rebuilding was performed using COOT (Emsley and Cowtan, 2004)and refinement using Refmac5 and PHENIX (Brünger et al., 1998; Adams et al.,2002). Statistics for both data collection and refinement are summarized inSupplemental Table S2.

Protein Pull-Down Assays

Pull-downexperimentswereperformed followingother authors (Igreja et al.,2014; Peter et al., 2015a, 2015b). In Figure 4, Cm eIF4G1003-1092-WT and mutantswere expressed with an N-terminal MBP tag in E. coli Rosetta (DE3) pLysS cellsas described above. The bacterial cells were resuspended in 2 mL of lysis bufferand lysed by sonication. A fixed concentration of purified Cm eIF4E (1 mM) wasadded to the MBP-tagged eIF4G1003-1092 (wild type and mutants) expressingcleared bacterial lysates, adjusted to 300 mL with lysis buffer (input fraction),and incubated with 30 mL of amylose resin (New England Biolabs) for 1 h at4°C. The beads werewashed three times with lysis buffer and eluted with 60mLof lysis buffer containing 25 mM maltose (bound fraction). To obtain roughlysimilar amounts of pulled-down Cm eIF4E, Cm eIF4G1003-1092 (wild type, C, NC,and C-NC) cleared lysates were incubated with purified Cm eIF4E in a ratio of3:1, 5:1, 2:1, and 4:1, respectively. Similarly, a cleared lysate of MBP-expressingbacteria was incubated with purified Cm eIF4E as negative control. Proteinswere analyzed by a 12% SDS-PAGE followed by Coomassie Brilliant Bluestaining. In Figure 6, MBP-tagged Cm eIF4G-WT and Cm eIF4E (wild typeandmutants) were expressed in E. coli Rosetta (DE3) pLysS cells as describedabove and cleared lysates were used. Pull-down assays were performedusing a 3:1 ratio of Cm eIF4G-WT and Cm eIF4E lysates (wild type andmutants). Eluted proteins were visualized by Coomassie Brilliant Blue Bluestaining and western blot (eIF4E) using a rabbit polyclonal antibody againstCm eIF4E (Miras et al., 2017b).

1488 Plant Physiol. Vol. 174, 2017

Miras et al.

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Binding Efficiency

For binding affinity experiments shown in Figure 5, cleared bacterial lysatesexpressing MBP-tagged wild-type and mutant Cm eIF4G-peptides (C, NC, andC-NC)were adjusted to the same recombinant protein concentration using lysisbuffer. Serial concentrations of 0.5, 1, 2, and 3 mM of purified Cm eIF4E wereadded to each Cm eIF4G lysate in a total volume of 300 mL and incubated withamylose resin for 1 h at 4°C. Protein elution and determination of eluted pro-teins were done as before. Band intensity of pulled-down Cm eIF4Es wasquantified with Quantity One software (Bio-Rad) and plotted versus total CmeIF4E input. Apparent dissociation constants were calculated using a one-sitebinding model (GraphPad Prism).

Translation Complementation by Transiently ExpressedCm eIF4E

Cm eIF4EH228 and mutant proteins were transiently expressed from binaryplasmids in cotyledons of melon (Cucumis melo) accession C46, which is ho-mozygous for Cm eIF4EH228L, as previously described (Nieto et al., 2006). At 3 to4 d postagroinfiltration, protoplasts were prepared from infiltrated tissues.In vivo translation assays were performed as previously described (Trunigeret al., 2008) by electroporating separately with two 59-UTR-luc-39-UTR RNAconstructs, differing in the UTRs flanking the luciferase gene that were eitherfrom isolate MNSV-Ma5 (eIF4E-dependent) or from the eIF4E-independentisolate MNSV-264. Translation of MNSV-264 can take place in protoplastsfrom eIF4E knocked-down lines, so it can function in the absence of eIF4E(Rodríguez-Hernández et al., 2012) and therefore serves for normalization ofthe different protoplast preparations. After 5 to 6 h incubation in the dark at25°C, protoplasts were lysed in 1x Passive lysis buffer (Promega). Firefly lucif-erase activities were measured with the Luciferase assay system (Promega). Foreach protoplast preparation, the translation efficiency determined for the con-struct of MNSV-264 was set to 100% and translation of MSNV-Ma5 wascompared to it. The expression levels of Cm eIF4E were analyzed in proteinextracts (extraction buffer: 0.1 M Tris HCl, pH 9.0, 0.1 M NaCl, 5 M urea,10 mM EDTA, 0.1 M b-mercaptoethanol) of infiltrated cotyledons and vi-sualized by western blot using a rabbit polyclonal antibody against CmeIF4E (Miras et al., 2017b).

Yeast Complementation

For the analysis of the complementation capacity of the Cm eIF4Emutants inJO55 yeast, the cDNAs encoding each of the Cm eIF4E variants were introducedinto the SpeI/BamHI restriction sites of the Trp-selectable yeast-E. coli shuttlevector p424-GDP/TRP1 (Mumberg et al., 1995) and used to transform JO55selected on Gal-containing minimal medium in the absence of Ura and Trp.After transformation, yeast cells were grown at 30°C in liquid Gal/Raf-Ura-Trpmedium until OD600 = 1, washed with sterile water, and serially diluted in10-fold steps until reaching 1,000-fold. Drops (5mL) of dilutionswere placed onboth control Gal/Raf-Ura-Trp solid medium and nitrogen base medium con-taining 2% Glc in the absence of Ura/Trp. JO55 transformed with an emptyp424-GPD/TRP1 vector was used as the negative control. Positive controlsincluded JO55 transformed with Arabidopsis thaliana eIF4E gene present invector p424-GPD/TRP1:At-eIF4E (Charron et al., 2008).

Accession Numbers

Coordinates for the structures described in this study have been depositedin the Protein Data Bank under the following accession numbers: 5ME5,Cm eIF4E:eIF4G1003-1092; 5ME6, Cm eIF4E:m7GDP; and 5ME7, Cm eIF4E.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. The canonical and NC eIF4E-binding domain ineIF4G and eIFiso4G.

Supplemental Figure S2. Fo-Fc difference map obtained after molecularreplacement using the C. melo eIF4E crystal structure as the search model.

Supplemental Figure S3. Structural comparisons of the melon, metazoan,and fungal eIF4G peptides bound to eIF4E.

Supplemental Figure S4. Sequence alignment of eIF4E orthologousproteins.

Supplemental Figure S5. Sequence alignment of plant eIF4Gs (top) andLOX2 proteins (bottom).

Supplemental Figure S6. Complementation of translation by Cm eIF4Emutants in eIF4E-deficient yeast.

ACKNOWLEDGMENTS

We thank Mari Carmen Montesinos and Blanca Gosalvez for their excellenttechnical assistance. Mario Fon edited the manuscript.

Received February 8, 2017; accepted May 15, 2017; published May 18, 2017.

LITERATURE CITED

Adams PD, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ,Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Terwilliger TC(2002) PHENIX: building new software for automated crystallographicstructure determination. Acta Crystallogr D Biol Crystallogr 58: 1948–1954

Aitken CE, Lorsch JR (2012) A mechanistic overview of translation initia-tion in eukaryotes. Nat Struct Mol Biol 19: 568–576

Altmann M, Müller PP, Pelletier J, Sonenberg N, Trachsel H (1989) Amammalian translation initiation factor can substitute for its yeast ho-mologue in vivo. J Biol Chem 264: 12145–12147

Ashby JA, Stevenson CEM, Jarvis GE, Lawson DM, Maule AJ (2011)Structure-based mutational analysis of eIF4E in relation to sbm1 resis-tance to pea seed-borne mosaic virus in pea. PLoS One 6: e15873

Bah A, Vernon RM, Siddiqui Z, Krzeminski M, Muhandiram R, Zhao C,Sonenberg N, Kay LE, Forman-Kay JD (2015) Folding of an intrinsicallydisordered protein by phosphorylation as a regulatory switch. Nature519: 106–109

Browning KS (2004) Plant translation initiation factors: it is not easy to begreen. Biochem Soc Trans 32: 589–591

Browning KS, Bailey-Serres J (2015) Mechanism of cytoplasmic mRNAtranslation. Arabidopsis Book 13: e0176

Brünger AT, Adams PD, Rice LM (1998) Recent developments for the ef-ficient crystallographic refinement of macromolecular structures. CurrOpin Struct Biol 8: 606–611

Charron C, Nicolaï M, Gallois JL, Robaglia C, Moury B, Palloix A, Caranta C(2008) Natural variation and functional analyses provide evidence forco-evolution between plant eIF4E and potyviral VPg. Plant J 54: 56–68

Collaborative CP; Collaborative Computational Project, Number 4 (1994)The CCP4 suite: programs for protein crystallography. Acta CrystallogrD Biol Crystallogr 50: 760–763

Dinkova TD, Martinez-Castilla L, Cruz-Espíndola MA (2016) The diver-sification of eIF4E family members in plants and their role in the plant-virus interaction. In G Hernández, R Jagus, eds, Evolution of the ProteinSynthesis Machinery and Its Regulation. Springer International Pub-lishing, Cham, Switzerland, pp 187–205

Emsley P, Cowtan K (2004) Coot: model-building tools for moleculargraphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132

Eskelin K, Hafrén A, Rantalainen KI, Mäkinen K (2011) Potyviral VPgenhances viral RNA translation and inhibits reporter mRNA translationin planta. J Virol 85: 9210–9221

Freire MA (2005) Translation initiation factor (iso) 4E interacts with BTF3,the b subunit of the nascent polypeptide-associated complex. Gene 345:271–277

Freire MA, Tourneur C, Granier F, Camonis J, El Amrani A, BrowningKS, Robaglia C (2000) Plant lipoxygenase 2 is a translation initiationfactor-4E-binding protein. Plant Mol Biol 44: 129–140

Gallie DR, Browning KS (2001) eIF4G functionally differs from eIFiso4G inpromoting internal initiation, cap-independent translation, and trans-lation of structured mRNAs. J Biol Chem 276: 36951–36960

Gao Z, Johansen E, Eyers S, Thomas CL, Noel Ellis TH, Maule AJ (2004)The potyvirus recessive resistance gene, sbm1, identifies a novel role fortranslation initiation factor eIF4E in cell-to-cell trafficking. Plant J 40:376–385

German-Retana S, Walter J, Doublet B, Roudet-Tavert G, Nicaise V,Lecampion C, Houvenaghel MC, Robaglia C, Michon T, Le Gall O

Plant Physiol. Vol. 174, 2017 1489

eIF4E Structure in Complex with an eIF4G Peptide

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

(2008) Mutational analysis of plant cap-binding protein eIF4E revealskey amino acids involved in biochemical functions and potyvirus in-fection. J Virol 82: 7601–7612

Gosselin P, Oulhen N, Jam M, Ronzca J, Cormier P, Czjzek M, Cosson B(2011) The translational repressor 4E-BP called to order by eIF4E: newstructural insights by SAXS. Nucleic Acids Res 39: 3496–3503

Gross JD, Moerke NJ, von der Haar T, Lugovskoy AA, Sachs AB,McCarthy JEG, Wagner G (2003) Ribosome loading onto the mRNA capis driven by conformational coupling between eIF4G and eIF4E. Cell115: 739–750

Grüner S, Peter D, Weber R, Wohlbold L, Chung M-Y, Weichenrieder O,Valkov E, Igreja C, Izaurralde E (2016) The structures of eIF4E-eIF4Gcomplexes reveal an extended interface to regulate translation initiation.Mol Cell 64: 467–479

Hinnebusch AG (2011) Molecular mechanism of scanning and start codonselection in eukaryotes. Microbiol Mol Biol Rev 75: 434–467

Hinnebusch AG, Lorsch JR (2012) The mechanism of eukaryotic translationinitiation: new insights and challenges. Cold Spring Harb Perspect Biol 4: 4

Igreja C, Peter D, Weiler C, Izaurralde E (2014) 4E-BPs require non-canonical 4E-binding motifs and a lateral surface of eIF4E to represstranslation. Nat Commun 5: 4790

Jackson RJ, Hellen CUT, Pestova TV (2010) The mechanism of eukaryotictranslation initiation and principles of its regulation. Nat Rev Mol CellBiol 11: 113–127

Kang BC, Yeam I, Frantz JD, Murphy JF, Jahn MM (2005) The pvr1 locusin Capsicum encodes a translation initiation factor eIF4E that interactswith Tobacco etch virus VPg. Plant J 42: 392–405

Kanyuka K, Druka A, Caldwell DG, Tymon A, McCallum N, Waugh R,Adams MJ (2005) Evidence that the recessive bymovirus resistance locusrym4 in barley corresponds to the eukaryotic translation initiation factor4E gene. Mol Plant Pathol 6: 449–458

Khan MA, Miyoshi H, Gallie DR, Goss DJ (2008) Potyvirus genome-linked protein, VPg, directly affects wheat germ in vitro translation:interactions with translation initiation factors eIF4F and eIFiso4F. J BiolChem 283: 1340–1349

Kinkelin K, Veith K, Grünwald M, Bono F (2012) Crystal structure of aminimal eIF4E-Cup complex reveals a general mechanism of eIF4Eregulation in translational repression. RNA 18: 1624–1634

Lax SR, Lauer SJ, Browning KS, Ravel JM (1986) Purification and prop-erties of protein synthesis initiation and elongation factors from wheatgerm. Methods Enzymol 118: 109–128

Léonard S, Viel C, Beauchemin C, Daigneault N, Fortin MG, Laliberté J-F(2004) Interaction of VPg-Pro of turnip mosaic virus with the translationinitiation factor 4E and the poly(A)-binding protein in planta. J GenVirol 85: 1055–1063

Lukhele S, Bah A, Lin H, Sonenberg N, Forman-Kay JD (2013) Interactionof the eukaryotic initiation factor 4E with 4E-BP2 at a dynamic bipartiteinterface. Structure 21: 2186–2196

Mader S, Lee H, Pause A, Sonenberg N (1995) The translation initiationfactor eIF-4E binds to a common motif shared by the translation factoreIF-4 gamma and the translational repressors 4E-binding proteins. MolCell Biol 15: 4990–4997

Marcotrigiano J, Gingras AC, Sonenberg N, Burley SK (1997) Cocrystalstructure of the messenger RNA 59 cap-binding protein (eIF4E) bound to7-methyl-GDP. Cell 89: 951–961

Marcotrigiano J, Gingras AC, Sonenberg N, Burley SK (1999) Cap-dependent translation initiation in eukaryotes is regulated by a molec-ular mimic of eIF4G. Mol Cell 3: 707–716

Matsuo H, Li H, McGuire AM, Fletcher CM, Gingras A-C, Sonenberg N,Wagner G (1997) Structure of translation factor eIF4E bound to m7GDPand interaction with 4E-binding protein. Nat Struct Biol 4: 717–724

Mayberry LK, Allen ML, Nitka KR, Campbell L, Murphy PA, BrowningKS (2011) Plant cap-binding complexes eukaryotic initiation factorseIF4F and eIFISO4F: molecular specificity of subunit binding. J BiolChem 286: 42566–42574

Miras M, Miller WA, Truniger V, Aranda MA (2017a) Non-canonicaltranslation in plant RNA viruses. Front Plant Sci 8: 494

Miras M, Truniger V, Querol-Audi J, Aranda MA (2017b) Analysis of theinteracting partners eIF4F and 39-CITE required for Melon necrotic spotvirus cap-independent translation. Mol Plant Pathol 18: 635–648

Miyoshi H, Okade H, Muto S, Suehiro N, Nakashima H, Tomoo K,Natsuaki T (2008) Turnip mosaic virus VPg interacts with Arabidopsisthaliana eIF(iso)4E and inhibits in vitro translation. Biochimie 90: 1427–1434

Mizuno A, In Y, Fujita Y, Abiko F, Miyagawa H, Kitamura K, Tomoo K,Ishida T (2008) Importance of C-terminal flexible region of 4E-bindingprotein in binding with eukaryotic initiation factor 4E. FEBS Lett 582:3439–3444

Monzingo AF, Dhaliwal S, Dutt-Chaudhuri A, Lyon A, Sadow JH,Hoffman DW, Robertus JD, Browning KS (2007) The structure of eu-karyotic translation initiation factor-4E from wheat reveals a novel di-sulfide bond. Plant Physiol 143: 1504–1518

Mumberg D, Müller R, Funk M (1995) Yeast vectors for the controlledexpression of heterologous proteins in different genetic backgrounds.Gene 156: 119–122

Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromo-lecular structures by the maximum-likelihood method. Acta CrystallogrD Biol Crystallogr 53: 240–255

Nicaise V, Gallois JL, Chafiai F, Allen LM, Schurdi-Levraud V, BrowningKS, Candresse T, Caranta C, Le Gall O, German-Retana S (2007) Co-ordinated and selective recruitment of eIF4E and eIF4G factors for po-tyvirus infection in Arabidopsis thaliana. FEBS Lett 581: 1041–1046

Nieto C, Morales M, Orjeda G, Clepet C, Monfort A, Sturbois B,Puigdomènech P, Pitrat M, Caboche M, Dogimont C, et al (2006) AneIF4E allele confers resistance to an uncapped and non-polyadenylatedRNA virus in melon. Plant J 48: 452–462

O’Brien JP, Mayberry LK, Murphy PA, Browning KS, Brodbelt JS (2013)Evaluating the conformation and binding interface of cap-binding pro-teins and complexes via ultraviolet photodissociation mass spectrome-try. J Proteome Res 12: 5867–5877

Paku KS, Umenaga Y, Usui T, Fukuyo A, Mizuno A, In Y, Ishida T,Tomoo K (2012) A conserved motif within the flexible C-terminus of thetranslational regulator 4E-BP is required for tight binding to the mRNAcap-binding protein eIF4E. Biochem J 441: 237–245

Papadopoulos E, Jenni S, Kabha E, Takrouri KJ, Yi T, Salvi N, Luna RE,Gavathiotis E, Mahalingam P, Arthanari H, et al (2014) Structure of theeukaryotic translation initiation factor eIF4E in complex with 4EGI-1 revealsan allosteric mechanism for dissociating eIF4G. Proc Natl Acad Sci USA 111:E3187–E3195

Park EH, Walker SE, Lee JM, Rothenburg S, Lorsch JR, Hinnebusch AG(2011) Multiple elements in the eIF4G1 N-terminus promote assembly ofeIF4G1cPABP mRNPs in vivo. EMBO J 30: 302–316

Patrick RM, Browning KS (2012) The eIF4F and eIFiso4F complexes ofplants: an evolutionary perspective. Comp Funct Genomics 2012: 287814

Pestova TV, Kolupaeva VG, Lomakin IB, Pilipenko EV, Shatsky IN, AgolVI, Hellen CUT (2001) Molecular mechanisms of translation initiation ineukaryotes. Proc Natl Acad Sci USA 98: 7029–7036

Peter D, Igreja C, Weber R, Wohlbold L, Weiler C, Ebertsch L,Weichenrieder O, Izaurralde E (2015a) Molecular architecture of 4E-BPtranslational inhibitors bound to eIF4E. Mol Cell 57: 1074–1087

Peter D, Weber R, Köne C, Chung M-Y, Ebertsch L, Truffault V,Weichenrieder O, Igreja C, Izaurralde E (2015b) Mextli proteins useboth canonical bipartite and novel tripartite binding modes to formeIF4E complexes that display differential sensitivity to 4E-BP regulation.Genes Dev 29: 1835–1849

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, MengEC, Ferrin TE (2004) UCSF Chimera–a visualization system for explor-atory research and analysis. J Comput Chem 25: 1605–1612

Rantalainen KI, Uversky VN, Permi P, Kalkkinen N, Dunker AK,Mäkinen K (2008) Potato virus A genome-linked protein VPg is an in-trinsically disordered molten globule-like protein with a hydrophobiccore. Virology 377: 280–288

Rodríguez-Hernández AM, Gosalvez B, Sempere RN, Burgos L, ArandaMA, Truniger V (2012) Melon RNA interference (RNAi) lines silencedfor Cm-eIF4E show broad virus resistance. Mol Plant Pathol 13: 755–763

Ruffel S, Gallois JL, Lesage ML, Caranta C (2005) The recessive potyvirusresistance gene pot-1 is the tomato orthologue of the pepper pvr2-eIF4Egene. Mol Genet Genomics 274: 346–353

Sanfaçon H (2015) Plant translation factors and virus resistance. Viruses 7:3392–3419

Siddiqui N, Tempel W, Nedyalkova L, Volpon L, Wernimont AK,Osborne MJ, Park H-W, Borden KLB (2012) Structural insights into theallosteric effects of 4EBP1 on the eukaryotic translation initiation factoreIF4E. J Mol Biol 415: 781–792

Sonenberg N, Hinnebusch AG (2009) Regulation of translation initia-tion in eukaryotes: mechanisms and biological targets. Cell 136: 731–745

1490 Plant Physiol. Vol. 174, 2017

Miras et al.

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.

Stein N, Perovic D, Kumlehn J, Pellio B, Stracke S, Streng S, Ordon F,Graner A (2005) The eukaryotic translation initiation factor 4E confersmultiallelic recessive Bymovirus resistance in Hordeum vulgare (L.).Plant J 42: 912–922

Truniger V, Aranda MA (2009) Recessive resistance to plant viruses. AdvVirus Res 75: 119–159

Truniger V, Nieto C, González-Ibeas D, Aranda M (2008) Mechanism ofplant eIF4E-mediated resistance against a Carmovirus (Tombusviridae):cap-independent translation of a viral RNA controlled in cis by an (a)virulencedeterminant. Plant J 56: 716–727

Umenaga Y, Paku KS, In Y, Ishida T, Tomoo K (2011) Identification andfunction of the second eIF4E-binding region in N-terminal domain ofeIF4G: comparison with eIF4E-binding protein. Biochem Biophys ResCommun 414: 462–467

Volpon L, Osborne MJ, Topisirovic I, Siddiqui N, Borden KL (2006) Cap-free structure of eIF4E suggests a basis for conformational regulation byits ligands. EMBO J 25: 5138–5149

Yeam I, Cavatorta JR, Ripoll DR, Kang BC, Jahn MM (2007) Functionaldissection of naturally occurring amino acid substitutions in eIF4E thatconfers recessive potyvirus resistance in plants. Plant Cell 19: 2913–2928

Plant Physiol. Vol. 174, 2017 1491

eIF4E Structure in Complex with an eIF4G Peptide

www.plantphysiol.orgon March 2, 2020 - Published by Downloaded from Copyright © 2017 American Society of Plant Biologists. All rights reserved.