Switch of the interactions between the ribosomal stalk and EF1A in the GTP- and GDP-bound conformations

Translation elongation factor EF1A delivers aminoacyl-tRNA to the ribosome in a GTP-bound form, and is released from the ribosome in a GDP-bound form. This association/dissociation cycle proceeds efficiently via a marked conformational change in EF1A. EF1A function is dependent on the ribosomal “stalk” protein of the ribosomal large subunit, although the precise mechanism of action of the stalk on EF1A remains unclear. Here, we clarify the binding mode of archaeal stalk aP1 to GTP-bound aEF1A associated with aPelota. Intriguingly, the C-terminal domain (CTD) of aP1 binds to aEF1A•GTP with a similar affinity to aEF1A•GDP. We have also determined the crystal structure of the aP1-CTD•aEF1A•GTP•aPelota complex at 3.0 Å resolution. The structure shows that aP1-CTD binds to a space between domains 1 and 3 of aEF1A. Biochemical analyses show that this binding is crucial for protein synthesis. Comparison of the structures of aP1-CTD•aEF1A•GTP and aP1-CTD•aEF1A•GDP demonstrates that the binding mode of aP1 changes markedly upon a conformational switch between the GTP- and GDP-bound forms of aEF1A. Taking into account biochemical data, we infer that aP1 employs its structural flexibility to bind to aEF1A before and after GTP hydrolysis for efficient protein synthesis.

domains 1 and 3, to which aP1-CTD binds in aP1•aEF1A•GDP (open conformation), is completely disrupted. Therefore, the stalk is expected to bind to aEF1A•GTP in a completely different mode to that when it is bound to aEF1A•GDP. However, no data is available on the structure of the aP1•aEF1A•GTP complex.
Elucidation of the molecular mechanism of binding between the stalk and aEF1A•GTP is important, because it would provide evidence for the action of the ribosomal stalk in recruitment to the ribosome of aEF1A•GTP associated with ligands (including aminoacyl-tRNA, aRF1, and aPelota), and also in the coupled GTP hydrolysis. Using archaeal samples, we here describe X-ray crystal structural analysis of the complex of aP1•aEF1A•GTP. We use a 17-mer peptide as an aP1-CTD sample and aPelota as a ligand of aEF1A to stabilize the GTP-bound state, and have determined the complex structure at 3.0 Å resolution. The structure shows that aP1-CTD binds to a hydrophobic cleft between domains 1 and 3 formed uniquely in the GTP-bound form of aEF1A. Moreover, the interactions between aP1-CTD and aEF1A•GTP seen in the crystal structure are evaluated by biochemical analysis using aEF1A mutants. The results suggest that, despite the large conformational change of aEF1A before and after GTP hydrolysis, interaction between aP1-CTD and aEF1A is maintained by switching of binding modes.

Results
Binding of aP1 to GTP-bound form of aEF1A. The ribosomal stalk protein aP1 promotes translation by recruiting aEF1A•GTP associated with its ligand (aminoacyl-tRNA, aRF1, or aPelota) to the ribosome. Therefore, we have analyzed the binding between aP1 and aEF1A•GTP in the presence of one of its ligands, aPelota. We initially attempted to prepare Pyrococcus horikoshii aPelota ( Pho Pelota), because we were able to show binding of Pho P1 to the GDP-bound form of elongation factor aEF1A using P. horikoshii Pho EF1A and elucidated the crystal structure of the complex 12 . Unfortunately, E. coli expression of Pho Pelota was poor. However, we found that Pyrococcus furiosus aPelota ( Pfu Pelota) was expressed at a sufficiently high enough level for the analysis. Furthermore, there is high homology in amino acid sequences between Pho Pelota and Pfu Pelota (Fig. S1). Therefore, we used Pfu Pelota with Pho P1 and Pho EF1A for further biochemical binding assays.
Our previous study showed that Pho P1 binds to the Pho EF1A•GTP•aminoacyl-tRNA complex via its C-terminal region 12 . Therefore, we tested whether the C-terminal region of Pho P1 binds to the Pho EF1A•GTP• Pfu Pelota complex, using a pull-down assay with maltose-binding protein fused to the C-terminal 14 amino acid sequence of Pho P1 (MBP-Pho P1C14). As shown in Fig. 1A, MBP-Pho P1C14 co-precipitated not only with Pho EF1A•GTP In vitro pull-down assay using amylose resin. Pho EF1A, MBP-Pho P1C14, and Pfu Pelota indicated at the top of the gel were mixed without (lanes 1-6) or with amylose resin (lanes 7-12). A portion of input samples (lanes 1-6) and protein samples bound to amylose resin (lanes 7-12) were subjected to SDS-PAGE. The proteins were detected by CBB staining. For reference, the uncropped gel image is included as (lane 10), but also with both Pho EF1A•GTP and Pfu Pelota (lane 12). However, Pfu Pelota was not precipitated in the sample lacking Pho EF1A•GTP (lane 11). The results indicate that the C-terminal region of Pho P1 binds to Pho EF1A within the Pho EF1A•GTP• Pfu Pelota complex. The binding between the C-terminal region and the Pho EF1A•GTP• Pfu Pelota complex was also detected by fluorescent polarization using a C-terminal 14-mer peptide of Pho P1, which was labeled with fluorescein isothiocyanate (FITC) at its N-terminus (Fig. 1B). Effective binding was not detected with a 11-mer peptide lacking the C-terminal 3 amino acids of the 14-mer peptide ( Pho P1C14∆3). The Kd value for the binding of the C-terminal peptide to the Pho EF1A•GTP• Pfu Pelota complex was estimated as 23.6 μM, a value which is comparable to the value of 10.4 μM determined for binding of the same C-terminal peptide to the Pho EF1A•GDP complex (Fig. 1C). The results indicate that the C-terminal region of Pho P1 binds both the Pho EF1A•GTP• Pfu Pelota and Pho EF1A•GDP complexes with a similar affinity.
Structural basis for interaction of aP1 stalk protein with aEF1A•Gtp•apelota complex. To elucidate the molecular details of the interaction between the C-terminal region of aP1 and the GTP-bound form of aEF1A, we attempted crystallization of the P1•EF1A•GTP•Pelota tetrameric complex using samples from several species including P. horikoshii, P. furiosus, and Aeropyrum pernix. The results of these efforts were a crystal of the A. pernix tetrameric complex Ape P1C17• Ape EF1A•GTP• Ape Pelota that diffracted to 3.0 Å resolution. The data collection and structure refinement statistics are summarized in Tables S1 and S2. The quality of the electron density maps surrounding aP1 and GTP in the complex is shown in Fig. S2. The overall structure of the tetrameric complex is shown in Fig. 2A. As reported by Kobayashi et al. 16 , the orientation of domains 1, 2, and 3 of Ape EF1A•GTP in the presence of Ape Pelota is that of the closed state (Fig. 2B), as observed in the tRNA-bound form of bacterial EF-Tu•GTP 14 . A part of the Ape P1C17 peptide (S103 to F111) was observed in a hydrophobic space between domains 1 and 3 in an extended unstructured form ( Fig. 2A). This P1 binding site was ~25 Å away from the GTP/GDP-binding site in domain 1.
Comparison of the structures of Ape P1C17-bound and Ape P1C17-free Ape EF1A•GTP• Ape Pelota revealed that, although the orientations of several amino acid side chains in the aP1 binding site of aEF1A differ markedly (details of the interaction between aEF1A and aP1 are described below), the overall structures of the two forms are highly similar (rms deviation of 0.49 Å for equivalent Cα atoms) (Fig. S3A,B). The conformations of regions including the P-loop and switch I and II elements, which are related to GTP hydrolysis, are also similar in the two structures (Fig. S3C). These results indicate that aP1 binding has no substantial effect on the overall structure of Ape EF1A including the GTP hydrolysis site in the domain 1.
Detailed interaction between aP1 and the GTP-bound form of aEF1A. The structure of the Ape P1C17• Ape EF1A•GTP• Ape Pelota complex revealed that the three hydrophobic amino acids, M108, F109, and F111 of Ape P1 make contact with several amino acid residues of Ape EF1A in the complex (Fig. 2C,D). Thus, M108 binds to R140 in domain 1 of Ape EF1A via van der Waals interaction; F109 binds to R329, F331, I421, and I423 in domain 3 of Ape EF1A via van der Waals interactions; F111 binds to R140 via hydrogen bonding, and to F401, R409, and I423 in domain 3 of Ape EF1A via van der Waals interactions. It is particularly noteworthy that the F109 residue of Ape P1, which is highly conserved among eukaryotes/archaea (Fig. S4), is surrounded by hydrophobic amino acids (F331, I421, and I423 in the case of Ape EF1A) (Fig. 2C), as is the case with the binding to aEF1A•GDP, aEF2•GMPPCP, aIF5B•GDP, and aABCE1•ADP 12,[19][20][21] . It is also notable that Ape P1 has an extra amino acid, F111, at its C-terminus, which is not present in other archaeal aP1 molecules (Fig. S4) and which does not seem to be essential for factor binding as described below.
Functional effect of mutations at the aEF1A•stalk binding sites. The crystal structural analysis indicates contact of M108, F109 and F111 of Ape P1 with Ape EF1A. The results are consistent with binding data shown in Fig. 1B, whereby deletion of the three C-terminal amino acids (L106, F107, and G108) of Pho P1 disrupted Pho EF1A binding. However, as described above, Ape P1 has an extra phenylalanine residue F111 at the C-terminus which is unique to Ape P1 (Fig. S4). We confirmed that deletion of F111 of Ape P1 resulted in no marked effect in binding to the Ape EF1A•GTP complex (Fig. S5). It is therefore likely that M108 and F109 of Ape P1 have the primary role in binding to Ape EF1A. Notably, the highly conserved F109 of Ape P1 binds to a hydrophobic pocket that is formed by the hydrophobic amino acids F331, I421, and I423 of Ape EF1A. To confirm the importance of this feature, we constructed a single mutant, F331A (the closest residue to F109 in Ape EF1A), and triple mutant F331A/I421A/I423 A (3A-mutant) of Ape EF1A, and tested their binding to Ape P1 (MBP-Ape P1C15) by pull-down assay (Fig. 3). Both the mutants associated with GTP and Ape Pelota showed a markedly reduced ability to bind to MBP-Ape P1C15 (lanes 9 and 10), compared with the wild type Ape EF1A (lane 8).
The present structural study showed that 7 amino acid residues (R140, R329, F331, F401, R409, I421, and I423) of Ape EF1A interact with the C-terminal region of Ape P1 (Fig. 2). Amino acid residues at these positions are conserved in archaea at least in terms of their properties (Fig. S6). Indeed, it is remarkable that R409 is highly conserved from archaea to eukaryotes. We next investigated the effect of mutations of these amino acid residues of EF1A on translation elongation. By using P. horikoshii elongation factors aEF1A/aEF2 with P. furiosus 70 S ribosomes, we have previously established a high temperature polyphenylalanine synthesis system 13 . Here we introduced six mutations in P. horikoshii EF1A, namely at R132, Q326, I328, V398, R406, and M420, which correspond to R140, R329, F331, F401, R409, and I423 in Ape EF1A, respectively. In the present crystal structure, all these amino acid residues interacts with Ape P1 via their side chains (A418 of Pho EF1A, which corresponds to I421 of Ape EF1A, was excluded from the mutagenesis study because it is an alanine residue). We then tested these mutants for poly(U)-dependent polyphenylalanine synthesis activity (Fig. 4). The R132A and I328S point mutations and I328S/M420S double mutation (which are presumed to interact with F107 of Pho P1, a highly conserved phenylalanine residue) caused considerable reduction in activity, while the M420S mutation resulted in only slightly reduced activity. In addition, the effects of these mutations of Pho EF1A were consistent with data on Ape EF1A in the Ape P1C17• Ape EF1A•GTP• Ape Pelota complex. Ape EF1A and Ape P1C17 are represented by a ribbon model, and the amino acid residues of Ape EF1A and Ape P1C17 that participate in the interaction by stick models. The color coding is the same as in (A). (D) Schematic diagram of the interactions between Ape P1C17 and Ape EF1A. The van der Waals contacts and hydrogen bonds are represented by black and red lines, respectively. Amino acid residues and numbering are for A. pernix samples, and those in parentheses are for P. horikoshii samples.

Discussion
The ribosomal stalk protein plays an important role in the recruitment of the GTP-bound form of elongation factor EF-Tu in bacteria, eEF1A in eukaryotes, and aEF1A in archaea, and thus in the delivery of the associated aminoacyl-tRNA to the A site of the ribosome. It had been expected that the stalk binding mode for GTP-bound aEF1A would differ from that for the GDP-bound form which has been reported previously 12 , because the relative location of domain 1 of aEF1A would be expected to differ in the closed GTP-bound and open GDP-bound conformations. In the present study using archaeal samples, we show that aP1-CTD binds to aEF1A•GTP with similar affinity to aEF1A•GDP. Furthermore, we have determined the crystal structure of the complex of the C-terminal aP1 peptide with aEF1A•GTP•aPelota, which has revealed that the binding mode of aP1 changes markedly upon the conformational switch between the closed and open conformations of aEF1A.
In the aP1-CTD•aEF1A•GTP•aPelota complex, an extended form of the aP1 peptide bound to the interface of domains 1 and 3 of aEF1A in the GTP-bound closed conformation (Fig. 2). This observation is supported by binding experiments (Fig. 3 and Table S3). All mutations of the putative aP1-binding site of aEF1A, which reduced the binding of aP1 to aEF1A•GTP, also decreased the ribosome-dependent polyphenylalanine synthesis (Fig. 4). Therefore, it is likely that the binding of aP1 to aEF1A•GTP as described in this study is functionally important and presumably contributes to recruitment of the ternary complex of aEF1A•GTP•aminoacyl-tRNA to the ribosome. It is interesting and worthwhile to consider the significance of why aP1 binds to the interface of domains 1 and 3 in aEF1A•GTP. A recent study using fluorescence resonance energy transfer (FRET) showed that EF-Tu•GTP is not locked in the closed conformation in solution, but adopts a dynamic conformation in which domain 1 can swing, even in the ternary complex with aminoacyl-tRNA 18 . Furthermore, this study also showed that the ribosomal factor binding center selectively accepts the EF-Tu•GTP•aminoacyl-tRNA complex in the closed conformation 18 . Based on these findings, we infer that aP1 facilitates the formation of the closed conformation of aEF1A•GTP by bridging domains 1 and 3, probably with the aid of aminoacyl-tRNA/aPelota/ aRF1, and promotes the selective binding of the closed-conformation aEF1A•GTP•aminoacyl-tRNA/aPelota/ aRF1 complex to the factor binding center of the ribosome.
To reinforce the idea that the binding of the C-terminal region of aP1 facilitates factor recruitment to the factor binding center of the ribosome, we constructed a docking model, using the structures of the bacterial Figure 3. Effect of mutations at F331, I421, and I423 of Ape EF1A, which were identified as the binding site of the conserved F109 of Ape P1. In vitro pull-down assay using amylose resin. Ape EF1A, MBP-Ape P1C15, and Ape Pelota indicated at the top of the gel were mixed without (lanes 1-5) or with amylose resin (lanes 6-10). A portion of input samples (lanes 1-5) and protein samples bound to amylose resin (lanes 6-10) were subjected to SDS-PAGE. The proteins were detected by CBB staining. For reference, the uncropped gel image is included as Fig. S9B. www.nature.com/scientificreports www.nature.com/scientificreports/ 70 S ribosome•EF-Tu•GDPCP•aminoacyl-tRNA complex 22 and the archaeal heptameric stalk protein complex 10 (Fig. 5). The model shows that the aP1-binding site within aEF1A is facing outward, and that the C-terminal region of aP1 can reach the ribosome-associated aEF1A•GTP via the flexible C-terminal half of aP1 without steric hindrance. Thus, this docking supports our view that aP1 directly binds to aEF1A via the C-terminal region of aP1, and contributes to the recruitment of aEF1A to the factor binding center of the ribosome.
Comparison of the current results describing aP1 binding to the GTP-bound form of aEF1A with previous results that describe binding to the GDP-bound form demonstrates that as expected the binding modes differ greatly in the two cases (Figs 6 and S7). In the results described here, the extended C-terminal peptide of aP1 binds to the interface of domains 1 and 3 of the closed GTP-bound conformation of aEF1A and this involves 3 residues of aP1 and 7 residues of aEF1A (Fig. S7). In the aP1•aEF1A•GDP, the C-terminal 27 residues of aP1 form an α-helix, and binds to a different site of the interface between domains 1 and 3 in the opened GDP-bound form of aEF1A, via 8 residues of aP1 and 9 residues of aEF1A (Fig. S7). Of these residues, only the conserved M108 and F109 of Ape P1 (L106 and F107 in Pho P1, respectively) are involved in binding of aP1 to both aEF1A•GDP and to aEF1A•GTP (Fig. S7). These findings suggest that the flexible C-terminal region of aP1 can bind to the two conformations of aEF1A in the process of GTP hydrolysis (aEF1A•GTP and aEF1A•GDP), and that it adopts an induced conformation dependent on the structures of aEF1A (Figs 6 and S8). This presumption is applicable to stalk binding to the other translational GTPases aIF5B 20 , aEF2 19 , and the translational ATPase aABCE1 21 . These crystal structural analyses have revealed that the conformation of the C-terminal region of aP1 in these complexes differs (Fig. S8). The variable structural properties of the C-terminal region which depends on associated proteins seems to be related to the functional role of the stalk proteins in efficient recruitment of various translational GTP/ATPases to the ribosome in translation.
The question arises as to why aP1 maintains the binding to switched GTP/GDP-bound conformations of aEF1A, and we propose a hypothesis as follows. As described above, the binding of aP1 to the GTP-bound form of aEF1A would promote the selective binding of the closed-conformation aEF1A•GTP•aminoacyl-tRNA/aPelota/aRF1 complex to the factor binding center of the ribosome. Upon the binding, GTP hydrolysis occurs and the conformational change of aEF1A from the closed conformation to the open conformation takes place on the ribosome. In this process, aP1 would temporarily release aEF1A, and subsequently aP1 could bind again to aEF1A to stabilize, in turn, the open GDP-bound conformation by bridging domains 1 and 3 in a different position from that in aEF1A•GTP (Figs 6 and S7). Because aEF1A•GDP is released form the ribosome in the open conformation, this stabilization of the open conformation of aEF1A by aP1 would promote the release of aEF1A•GDP from the ribosome. Furthermore, we infer that the switching of the binding of the stalk mentioned above also takes place in eukaryotes (eEF1A•GTP/GDP) and eubacteria (EF-Tu•GTP/GDP). Further studies are needed to validate these hypotheses.
Methods plasmid construction. The coding sequences for elongation factor aEF1A of the hyperthermophilic archaeon Aeropyrum pernix ( Ape EF1A) were amplified from the genome by PCR, and inserted between the NdeI and XhoI sites in the E. coli expression vector, pET-15b containing a six His-tag. The coding sequences for aPelota were amplified from Pyrococcus furiosus and A. pernix genomes, and inserted between the NdeI and BamHI sites in the pET-15b vector. The plasmid for expression of P. horikoshii aEF1A ( Pho EF1A) and aEF2 ( Pho EF2) were as described previously 12,23 . The plasmid for expression of N-terminal maltose binding protein (MBP) fused with www.nature.com/scientificreports www.nature.com/scientificreports/ the C-terminal 14 amino acid residues of P. horikoshii aP1 (MBP-Pho P1C14) was constructed by removing the coding sequence for residues 61-94 from pMAL-c4x-aP1[61-108] 21 by PCR. Although pMAL-c4x-aP1[61-108] was prepared using the P. furiosus genome, the amino acid sequence for the C-terminal 14 amino acid residues of P. furiosus aP1 is identical to that of P. horikoshii aP1 (Fig. S4). The plasmid for expression of N-terminal MBP fused with the C-terminal 15 amino acid residues of A. pernix aP1 (MBP-Ape P1C15) was constructed by insertion of the PCR-amplified sequence corresponding to the C-terminal 15 amino acid residues of A. pernix aP1 between the EcoRI and PstI sites in the pMAL-c4x vector. Site-directed mutagenesis of Pho EF1A or Ape EF1A was performed by PCR using the plasmid carrying each EF1A gene, as described previously 12 . The PCR primers are listed in Table S4.
Protein expression and purification. Ape EF1A, Pho EF1A, their mutants, Pfu Pelota, Ape Pelota, and the fusion proteins MBP-Pho P1C14 and MBP-Ape P1C15 were all expressed in E. coli BL21 (DE3) codon plus RIL or BL21 (DE3) by adding 0.5 mM IPTG. After incubation at 37 °C for 3 h (for Pho EF1A, its mutants, and MBP-Pho P1C14) or 20 °C for 18 h (for Ape EF1A, its mutants, Ape Pelota, Pfu Pelota, and MBP-Ape P1C15), cells were harvested by centrifugation, resuspended in Lysis buffer (20 mM HEPES-KOH pH7.6, 1 M NH 4 Cl, 5% (v/v) glycerol, 7 mM 2-ME) and disrupted by sonication. After sedimenting cell debris and other insoluble materials, Pho EF1A, Ape EF1A and their mutants were purified by heat treatment (80 °C for 20 min) of cell extracts, Ni-affinity chromatography and size-exclusion chromatography. The His-tag of aEF1A samples and its mutants was cleaved with thrombin before anion-exchange chromatography. To prepare the GTP-bound form of aEF1A, tightly bound GDP was removed from purified aEF1A by bacterial alkaline phosphatase treatment, as described previously 12 . MBP-Pfu P1C14 and MBP-Ape P1C15 were purified by amylose-affinity chromatography and size-extraction chromatography. Ape Pelota and Pfu Pelota were purified by heat treatment (70 °C for 20 min) of cell extracts, Ni-affinity chromatography and anion-exchange chromatography.