Title: Nmd3 is a structural mimic of <scp>eIF</scp> 5A, and activates the cp <scp>GTP</scp> ase Lsg1 during 60S ribosome biogenesis
Abstract: Article8 February 2017free access Source DataTransparent process Nmd3 is a structural mimic of eIF5A, and activates the cpGTPase Lsg1 during 60S ribosome biogenesis Andrey G Malyutin Andrey G Malyutin Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Sharmishtha Musalgaonkar Sharmishtha Musalgaonkar Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Stephanie Patchett Stephanie Patchett Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Joachim Frank Corresponding Author Joachim Frank [email protected] orcid.org/0000-0001-5449-6943 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Department of Biological Sciences, Columbia University, New York, NY, USA Howard Hughes Medical Institute, Columbia University, New York, NY, USA Search for more papers by this author Arlen W Johnson Corresponding Author Arlen W Johnson [email protected] orcid.org/0000-0002-4742-085X Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Andrey G Malyutin Andrey G Malyutin Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Search for more papers by this author Sharmishtha Musalgaonkar Sharmishtha Musalgaonkar Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Stephanie Patchett Stephanie Patchett Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Joachim Frank Corresponding Author Joachim Frank [email protected] orcid.org/0000-0001-5449-6943 Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA Department of Biological Sciences, Columbia University, New York, NY, USA Howard Hughes Medical Institute, Columbia University, New York, NY, USA Search for more papers by this author Arlen W Johnson Corresponding Author Arlen W Johnson [email protected] orcid.org/0000-0002-4742-085X Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA Search for more papers by this author Author Information Andrey G Malyutin1,‡, Sharmishtha Musalgaonkar2,‡, Stephanie Patchett2, Joachim Frank *,1,3,4 and Arlen W Johnson *,2 1Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA 2Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX, USA 3Department of Biological Sciences, Columbia University, New York, NY, USA 4Howard Hughes Medical Institute, Columbia University, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +1 212 305 9510; Fax: +1 212 305 9500; E-mail: [email protected] *Corresponding author. Tel: +1 512 475 6350; Fax: +1 512 471 7088; E-mail: [email protected] The EMBO Journal (2017)36:854-868https://doi.org/10.15252/embj.201696012 See also: A Razi & J Ortega (April 2017) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract During ribosome biogenesis in eukaryotes, nascent subunits are exported to the cytoplasm in a functionally inactive state. 60S subunits are activated through a series of cytoplasmic maturation events. The last known events in the cytoplasm are the release of Tif6 by Efl1 and Sdo1 and the release of the export adapter, Nmd3, by the GTPase Lsg1. Here, we have used cryo-electron microscopy to determine the structure of the 60S subunit bound by Nmd3, Lsg1, and Tif6. We find that a central domain of Nmd3 mimics the translation elongation factor eIF5A, inserting into the E site of the ribosome and pulling the L1 stalk into a closed position. Additional domains occupy the P site and extend toward the sarcin–ricin loop to interact with Tif6. Nmd3 and Lsg1 together embrace helix 69 of the B2a intersubunit bridge, inducing base flipping that we suggest may activate the GTPase activity of Lsg1. Synopsis Cryo-EM structures of the yeast 60S subunit bound to assembly factors Nmd3, Lsg1, and Tif6 reveal the conformational rearrangements that take place during final ribosome maturation in the cytoplasm. Structure of the 60S subunit export adapter Nmd3 and the GTPase Lsg1 at 3.1–4.5 Å resolution. Nmd3 spans the intersubunit interface of the 60S subunit from the L1 stalk through the E and P sites to Tif6. An eIF5A-like domain of Nmd3 induces closure of the L1 stalk. Nmd3 must retract from the P site to allow binding of Sdo1 and final maturation steps to occur. Activation of Lsg1 GTPase requires both Nmd3 and 60S subunits. Lsg1 binding to helix 69 causes base flipping of G2661. Introduction The ribosome is a remarkably intricate and dynamic machine whose assembly requires the precise processing and folding of its RNA along with the incorporation of numerous ribosomal proteins. Because the mature ribosome and its attendant translation factors are responsible for decoding the cell's genome during mRNA translation, the correct assembly of this complex machine is critical for faithful gene expression. The eukaryotic ribosome comprises a large (60S) and a small (40S) subunit. Their synthesis begins with rRNA transcription in the nucleolus where initial assembly of the SSU processome promotes co-transcriptional processing and folding to form the 90S particle (Dragon et al, 2002; Grandi et al, 2002; Woolford & Baserga, 2013; Gerhardy et al, 2014; Kornprobst et al, 2016). An early cleavage event releases the pre-40S from the assembling pre-60S subunit (Osheim et al, 2004; Koš & Tollervey, 2010; Karbstein, 2013; Woolford & Baserga, 2013; Gerhardy et al, 2014; Henras et al, 2015). The two subunits then follow independent paths of additional assembly events, nuclear export, and final maturation in the cytoplasm (Tschochner & Hurt, 2003; Zemp & Kutay, 2007; Panse & Johnson, 2010; Thomson et al, 2013). Export of the 60S subunit in eukaryotes is strictly dependent on Nmd3, a highly conserved protein whose C-terminal leucine-rich nuclear export signal recruits the export receptor Crm1 (Ho et al, 2000; Gadal et al, 2001; Thomas & Kutay, 2003; Trotta et al, 2003). Additional factors, including Arx1 and the mRNA export factor Mex67-Mtr2, also contribute to export in yeast (Bradatsch et al, 2007; Yao et al, 2007; Hung et al, 2008). Although hundreds of transiently interacting factors, many of which are essential proteins, orchestrate the cascade of assembly events from the nucleolus to the cytoplasm (Nissan et al, 2002; Fromont-Racine et al, 2003; Strunk & Karbstein, 2009; Kressler et al, 2010; Thomson et al, 2013; Woolford & Baserga, 2013), the majority of these factors are released prior to nuclear export (Gerhardy et al, 2014). Thus, the complexity of the pre-ribosomal particles that enter the cytoplasm is significantly reduced. The final maturation of 60S subunits in the cytoplasm involves the release and recycling of trans-acting factors and the incorporation of the remaining ribosomal proteins (reviewed in Zemp & Kutay, 2007; Panse & Johnson, 2010; Karbstein, 2013), resulting ultimately in their functional activation. These events can be ordered into a pathway that reveals a hierarchical dependence on ATPase- and GTPase-driven maturation steps (Lo et al, 2010). The final known steps in cytoplasmic maturation include assembly of the P stalk (Kemmler et al, 2009; Lo et al, 2009) and subsequent release of the ribosome anti-association factor, eIF6 (Tif6 in yeast), and the nuclear export factor Nmd3. The GTPase Efl1, a paralog of the translation elongation factor EF2, together with the protein Sdo1, promote the release of Tif6 (Senger et al, 2001; Weis et al, 2015). Because Tif6 sterically blocks association of the 40S subunit (Russell & Spremulli, 1980; Gartmann et al, 2010), it must be removed to allow assembly of translationally active 80S ribosomes (Raychaudhuri et al, 1984). We and others have proposed that this release event represents a quasi-functional “test drive” of the nascent 60S subunit in which protein mimics of translation factors assess functionality of newly made subunits prior to their release into the translating pool (Bussiere et al, 2012; Weis et al, 2015). Although Nmd3 functions in nuclear export, it remains associated with pre-60S particles until late in cytoplasmic maturation. Its release depends on the assembly of uL16 (Rpl10) into the subunit to complete the peptidyl-transferase center (Bussiere et al, 2012) and assembly of eL40 (Fernández-Pevida et al, 2012). In addition, depletion of Sdo1 or Efl1 leads to accumulation of both Tif6 and Nmd3 on subunits (Lo et al, 2010; Finch et al, 2011), suggesting that Nmd3 is released in concert with or after Tif6. Thus, at the time of release of Nmd3 by the GTPase Lsg1 (Hedges et al, 2005), all known assembly events on the 60S subunit have been completed. Although molecular genetic studies have identified the functions of many individual ribosome biogenesis factors and the order of events, the way these factors collaborate to assemble ribosomes has only begun to be unraveled by recent structural studies. High-resolution cryo-electron microscopy (EM) of native nuclear pre-ribosomal particles has revealed the architecture of several assembly intermediates, delineating the structures and binding sites for numerous trans-acting factors (Bradatsch et al, 2012; Bussiere et al, 2012; Greber et al, 2012; Leidig et al, 2014; Kornprobst et al, 2016; Murray et al, 2016; Wu et al, 2016). These structures display remarkable complexity of interactions among transacting factors and the pre-60S subunit, as well as unexpected insights to RNA rearrangements that must occur during ribosome assembly (reviewed in Greber, 2016). Among the factors on the nuclear pre-60S subunit is the essential nuclear GTPase Nog2 (also named Nug2) as well as numerous shuttling factors that are exported with the pre-60S subunit, including eIF6 and Arx1. Structures have also been determined for complexes of eIF6 and Arx1 reconstituted with mature 60S subunits (Klinge et al, 2011; Greber et al, 2016). However, thus far, these structures have lacked the export adaptor, Nmd3. UV cross-linking studies have suggested that Nmd3-rRNA contacts overlap with the Nog2 binding site (Matsuo et al, 2014). Thus, recruitment of Nmd3 is thought to be regulated by the release of Nog2 as a checkpoint for nuclear export (Matsuo et al, 2014). While the structures of these nuclear pre-ribosomal intermediates have provided important insights into assembly mechanisms and structural transitions that take place prior to export, less is known about the events of cytoplasmic maturation of pre-60S particles. However, recent cryo-EM of partially reconstituted cytoplasmic 60S with eIF6, Sdo1, and Efl1 identified Sdo1 as a ribosome recycling factor-like protein that binds in the P site to engage and activate the GTPase activity of Efl1 (Weis et al, 2015). Here, we have focused on the late cytoplasmic 60S-Nmd3-Lsg1 complex. Whereas many earlier ribosome biogenesis steps involve rearrangements of RNA and proteins that are not amenable to reconstitution, we reasoned that the 60S-Nmd3-Lsg1 complex, representing the completed subunit before release of Nmd3, could be faithfully reconstituted in vitro. We present a three-dimensional (3D) reconstruction of the yeast proteins Nmd3, Lsg1, and Tif6 in complex with the 60S subunit, determined by single-particle cryo-electron microscopy. 60S-Nmd3 and 60S-Nmd3-Lsg1-Tif6 complexes were resolved to 3.1 and 3.3 Å, respectively. Lsg1 is an active GTPase in this complex and requires the presence of both the 60S subunit and Nmd3. Thus, this reconstituted complex likely captures the salient features of Nmd3 and Lsg1 binding. Results Reconstitution of a 60S-Nmd3-Lsg1-Tif6 complex We reconstituted complexes of yeast 60S subunits with combinations of Nmd3, Lsg1, and Tif6 from purified components in vitro. Nmd3 was purified from yeast as a fusion to maltose-binding protein (MBP). Lsg1 was purified from E. coli with a C-terminal 6xHistidine tag. Lsg1 belongs to the family of circularly permuted GTPases (cpGTPases) in which the G motifs are reordered (Reynaud et al, 2005; Anand et al, 2006). Although it has previously been reported that free human Lsg1 is an active GTPase in vitro (Reynaud et al, 2005), this had not been shown for the yeast protein. We found that free yeast Lsg1 protein had no detectable GTP hydrolysis activity (Fig 1A). The GTPase activity was not stimulated by Nmd3 alone and only modestly stimulated by 60S subunits. However, in the presence of both 60S subunits and Nmd3, we observed robust GTPase activity (Fig 1A). Neither Nmd3 nor free 60S subunits had significant GTPase activity themselves, demonstrating that the increased GTP hydrolysis by Lsg1 resulted from the stimulation of the Lsg1 GTPase center by the 60S subunit and Nmd3 together, and was not an additive effect of the individual components. In the presence of Nmd3 and 60S subunits, a minimum estimate of the rate of GTP hydrolysis by Lsg1 was 190 min−1 (Appendix Fig S1), comparable to, but higher than, the turnover rate of 14 min−1 reported for the bacterial cpGTPase RbgA in the presence of 50S subunits (Achila et al, 2012). Unlike previously characterized cpGTPases (Achila et al, 2012; Ash et al, 2012; Matsuo et al, 2014), Lsg1 was also not stimulated by increased concentrations of potassium, either as free protein or in the presence of 60S and Nmd3 (Appendix Fig S1B). Together, these results demonstrate that the reconstituted 60S-Nmd3-Lsg1 complex is active. Figure 1. Lsg1 and Nmd3 form an active complex with the 60S subunit Percent GTP hydrolysis in reactions containing 125 nM Lsg1, 100 nM Nmd3, or 25 nM 60S alone, or in the indicated combinations, was determined by monitoring the release of free phosphate as described in Materials and Methods. Representative curves for percent GTP hydrolysis by increasing concentrations of Lsg1, as indicated in the figure, without Nmd3 or 60S subunit (green) or reactions containing 100 nM Nmd3 with 25 nM (black) and 50 nM (blue) 60S. Representative curves for percent GTP hydrolysis by 125 nM Lsg1 in reactions containing 25 nM (black) and 50 nM (blue) 60S subunits, and increasing concentrations of Nmd3. Migration of 60S-Nmd3-Lsg1 complex (stoichiometry of 1:4:5) in the presence of GMPPNP after sedimentation through 10–30% sucrose. Fractions were precipitated with 10% TCA and analyzed on 10% SDS–PAGE. MBP-(TEV)-HIS6-Nmd3 and Lsg1-6His and 60S subunit proteins are indicated. Data information: Percent GTP hydrolysis values for Lsg1 and Nmd3 titrations in (B) and (C) were fitted to saturating-specific single-site binding curves using the GraphPad Prism software. All reactions were performed in triplicate. Bars indicate mean and standard deviation. Source data are available online for this figure. Source Data for Figure 1 [embj201696012-sup-0003-SDataFig1.tif] Download figure Download PowerPoint To ensure sufficient occupancy of Lsg1 and Nmd3 on 60S subunits for 3D reconstruction, we performed titration experiments, using the dependence of the Lsg1 GTPase on both Nmd3 and 60S subunits as a proxy for protein occupancy. We reasoned that monitoring Lsg1 GTPase activity would report on Lsg1 binding to a specific site on the 60S subunit and avoid issues that could arise from non-specific binding. We titrated Lsg1 at two different 60S subunit concentrations, keeping Nmd3 constant (Fig 1B). At each 60S concentration, GTP hydrolysis approached a maximum as Lsg1 concentration was increased, suggesting that Lsg1 approached saturation. Near-saturation with Lsg1 was achieved at ~10 Lsg1 molecules per 60S subunit. A similar titration of Nmd3 at constant 60S subunit concentration and two concentrations of Lsg1 showed saturation with Nmd3 at a ratio of ~4 Nmd3 molecules per 60S subunit (Fig 1C). GTP hydrolysis data in Fig 1B and C were fitted to saturating-specific single-site binding curves. Parabolic standard slope saturating curves indicated that both Lsg1 and Nmd3 bind to single sites in the 60S-Nmd3-Lsg1 complex. Sucrose density gradient sedimentation of 60S-Nmd3-Lsg1 complexes in the presence of the non-hydrolyzable GTP analog guanosine 5′-[β,γ-imido]triphosphate (GMPPNP) confirmed that 60S, Nmd3 and Lsg1 co-sedimented as a stable complex (Fig 1D). Structure determination of Nmd3-containing complexes To better understand the function of Nmd3 and Lsg1 in the assembly of the 60S subunit, we utilized cryo-EM to characterize the structures of 60S-Nmd3, 60S-Nmd3-Lsg1, and 60S-Lsg1 complexes. Lsg1-containing complexes were prepared in the presence of GMPPNP. To improve the distribution of particle orientations in ice, 0.5% w/v of glutaraldehyde was added to complexes prior to freezing. Refined maps for 60S-Nmd3 and 60S-Nmd3-Lsg1 complexes contained densities not present on the mature 60S subunits alone. However, 60S-Lsg1 maps showed no extra density compared to the mature 60S subunit. Consequently, we focused our effort on the 60S-Nmd3 and 60S-Nmd3-Lsg1 complexes. In the 60S-Nmd3 complex, a mass of density could be seen between the A site and the SRL, but only at low thresholds, suggesting a high degree of flexibility of this domain of Nmd3. This region was better defined in the presence of Lsg1 and appeared to project toward the position that Tif6 would occupy on the 60S subunit. Because Tif6 and Nmd3 are both present on the same pre-60S particles in vivo (Lo et al, 2010), we prepared and characterized an additional 60S-Nmd3-Lsg1-Tif6 (60SNLT) complex in order to determine if Nmd3 made contacts with Tif6. It was apparent that both Nmd3 and the L1 stalk existed in multiple conformations in these complexes, with the L1 stalk ranging from open to closed. In the closed and intermediate states, additional density could be seen in contact with the L1 stalk. To separate the various states and improve resolution of Nmd3 and Lsg1, we used 3D classification with signal subtraction (Bai et al, 2015). The best maps were achieved with the stalk in the closed state, primarily due to high representation of this state in our particles. A 60SNLT map was refined to 3.3 Å resolution from ~19,000 particles (Appendix Table S1). To further improve the quality of the Nmd3 density, datasets from 60S-Nmd3, 60S-Nmd3-Lsg1, and 60SNLT were combined into a single set after 3D classification of individual runs, resulting in a total of 226,516 particles. The combined 60S-Nmd3 dataset resulted in an overall map at 3.1 Å resolution, with interior 60S subunit regions reaching resolutions better than 3 Å (Appendix Fig S2). See Appendix Figs S3–S8 and Appendix Table S1 for classification and refinement strategies. Comparing the maps for 60S-Nmd3 and 60SNLT to that of a mature 60S subunit, we were able to assign densities not present on the mature 60S subunit to Nmd3, Lsg1, and Tif6 (Fig 2A). Although we reconstituted complexes using Nmd3 with an N-terminal MBP tag, we did not observe any density for MBP, indicating that MBP did not adopt any specific position in the reconstituted particle. To gain greater insight into the function of Nmd3 and its interaction with the subunit, we sought to assign amino acid sequence to the density. However, there were no structures for Nmd3-family proteins, and computer-generated models did not predict structures that corresponded to the observed density. Fortunately, the majority of Nmd3 density in the combined 60S-Nmd3 map that we observed between uL1 and the A site was of sufficiently high resolution to allow assignment of 254 amino acids, A147 to K401 (Figs 2A and C, and 3A). The 60SNLT complex was used to build a portion of the remaining N-terminal residues from 40 to 146. The C-terminal 117 amino acids of Nmd3, containing the nuclear import and export signals (Hedges et al, 2006), were not resolved in our structure. Figure 2. Structure of the 60S-Nmd3-Lsg1-Tif6-GMPPNP complex Cryo-EM reconstruction of yeast 60S subunit (white) in complex with Nmd3 (orange), Lsg1 (green), and Tif6 (yellow). A site, P site, and E site and regions of the density map shown in detail in panels (C) and (D) are indicated. Zoomed view of reconstruction from a class of particles with a deformed H38 (shown in blue). The elbow region of Nmd3 connecting the eL22-like and N-terminal domains and an unassigned density connecting Lsg1 and Nmd3 are also shown. Region extracted from density map in (A) showing the interface region of Nmd3 and eL42. Region extracted from density map in (A) showing density for Lsg1 wedged underneath uL14. Data information: Density contours in (C) and (D) are shown at 4 sigma and Pymol carve level of 2.2 Å. Download figure Download PowerPoint Figure 3. The structure of Nmd3 Linear map of Nmd3 and atomic structure colored on proposed domains. Amino acid positions are given by numbers. Sequence and structural alignment of eIF5a (golden) with eIF5a and eL22-like domains of Nmd3. Comparison of Nmd3 from the 60S-Nmd3-Lsg1 complex (upper panel, colored as in A) and eIF5A (PDB: 5gak) (lower panel, golden) bound to the 60S subunit. The L1 stalk (light blue), uL1 (pink), and eL42 (green) are highlighted. Download figure Download PowerPoint Nmd3 spans the joining face of the 60S subunit from Tif6 to the L1 stalk In both 60S-Nmd3 and 60SNLT complexes, the primary state for the 60S subunit was with the L1 stalk in the closed conformation and Nmd3 spanning the 60S subunit from the uL1 protein on the L1 stalk through the E site, the P site and extending toward Tif6 at the sarcin–ricin loop (SRL). The position of Nmd3 in our reconstituted particles was remarkably similar to that of unidentified densities in the recently described structure of a Yvh1-containing pre-60S particle (Sarkar et al, 2016). In that work, density attributed to Nmd3 was observed spanning from Tif6 to the P site, while additional density between the L1 stalk and the P site was tentatively attributed to Lsg1. Based on the structure presented here, we can attribute both of the unassigned densities in the Yvh1-particle to Nmd3 alone. Overall, the assigned Nmd3 sequence can be separated into three domains (Fig 3A). The N-terminal domain is composed of a long four-stranded beta sheet and two alpha helices, and spans from Tif6 toward the P site (SRL; helices 89, 91, 92, and 95). Although the extreme N-terminus of Nmd3 (residues 1–39) was not of sufficient resolution to assign amino acids, this region of Nmd3 appears to interact directly with Tif6 (Fig 2A). Possibly, the MBP fusion destabilizes the Nmd3-Tif6 interaction, leading to reduced resolution of the extreme N-terminus of Nmd3. Because we could resolve the N-terminal domain of Nmd3 only in the presence of Tif6, the interaction of Tif6 appears to stabilize the N-terminus of Nmd3. Consistent with this stabilization, the addition of Tif6 modestly enhanced the Nmd3-stimulated Lsg1 GTPase activity (Appendix Fig S1C). The second domain of Nmd3, spanning residues 152 through 255, is composed of a five-stranded beta sheet and two alpha helices, and contains a long flexible loop (Fig 3B). This domain occupies the ribosomal P site in the closed state of the L1 stalk. In this state, the flexible loop (V229–V240) is positioned directly above and covering the peptide exit tunnel. We compared the fold of this domain to known structures using the DALI server and found that it adopts a similar fold as that of ribosomal protein eL22 (Z = 5.5, r.m.s.d. 3.0 Å, PDB: 4uk1, chain H). Thus, we refer to this domain as eL22-like. Despite the similarity of folds, we do not suggest functional relatedness between eL22 and Nmd3. At the elbow-like junction of the N-terminal and eL22-like domains, Nmd3 closely approaches the peptidyl (P) site loop of uL16 (Fig 2B). This flexible loop of uL16 is required for the release of Nmd3 from the nascent subunit (Bussiere et al, 2012). Mutations at the base of this loop (R98S, R98C, H123P) are associated with T-cell acute lymphoblastic leukemia in humans, and they also impair the release of Nmd3 and Tif6 (De Keersmaecker et al, 2013). This loop of uL16 is stabilized by P site ligands but is unstructured in our model. Extension of the loop into the P site, as observed for mature ribosomes in the presence of tRNA or eIF5A (Schmidt et al, 2016), would be precluded in our structure due to steric clash with Nmd3. As a consequence, the P site loop of uL16 is not resolved and must be displaced above the elbow region at A147 of Nmd3. The third domain of Nmd3 (aa 256–401) interacts with uL1 of the L1 stalk and eL42. Thirty of the assigned amino acids of Nmd3 are at the interface with uL1, resulting in a buried surface area of ~577 Å2 (Fig 3C). However, due to poor resolution of uL1 density, assignment of amino acids of uL1 could not be performed reliably. Nmd3 shares an interface of 744 Å2 with eL42, composed of 24 residues of Nmd3 and 21 residues of eL42 (Figs 2C and 3C). This domain of Nmd3 interface is stabilized by a number of predicted hydrogen bonds and salt bridges (Appendix Table S2; PDBePISA server). A domain of NMD3 resembles eIF5A in both structure and mode of binding Euryarchaeal Nmd3 proteins have previously been annotated as containing an eIF5A-like domain, based on sequence comparison (Aravind & Koonin, 2000). However, no sequence similarity between eukaryotic Nmd3 and eIF5A proteins had been reported. Our structure shows that the third domain of Nmd3 (aa 256–401) adopts the same topology as that of eIF5A (Dali: Z = 3.5, r.m.s.d 4.0 Å, PDB: 5gak-q). In addition, we detected limited sequence similarity between residues 289–326 of Nmd3 and 66–97 of eIF5A (Fig 3B). We will refer to this domain as the eIF5A-like domain of Nmd3. eIF5A and its bacterial homolog EF-P are necessary for the rescue of ribosomes stalled on polyproline-containing sequences and function by recognizing an empty E site (Doerfel et al, 2013; Gutierrez et al, 2013; Ude et al, 2013). eIF5A is located in the tRNA path of the 60S subunit between the E and P sites (Melnikov et al, 2016). eIF5A interacts with uL1, inducing a closed conformation of the L1 stalk (Schmidt et al, 2016). This closed conformation is additionally stabilized by interactions between the N-terminal extension of eIF5A and eL42 (Fig 3C, lower panel). The position of Nmd3 in the tRNA path, its interaction with eL42 and uL1, and its influence on inducing a closed position of the L1 stalk are remarkably similar to the interaction of eIF5A with the 60S subunit (Fig 3C, Movie EV1). An essential element of eIF5A is the extended β3–β4 loop that carries hypusine and interacts with the acceptor stem of P site tRNAs (Wolff et al, 2007). The corresponding loop in Nmd3 (determined by structural alignment of eIF5a-like domain and eIF5A) is severely truncated, lacks the corresponding lysine of eIF5A that is modified to hypusine, and does not protrude beyond the core of the domain. eIF5A contains an N-terminal extension that emanates from a tight turn and folds back along the protein toward the L1 stalk (Melnikov et al, 2016; Schmidt et al, 2016). In Nmd3, the corresponding strand projects forward, toward the P site, and contributes to the five-stranded beta sheet in the eL22-like domain. Interestingly, this strand supports a flexible loop (aa 229–239) that extends beyond the core of the eL22-like domain, reminiscent of the hypusine-containing β3–β4 loop of eIF5A. However, this loop of Nmd3 does not contain a lysine that could be a substrate for modification. Nmd3 and the L1 stalk exhibit multiple conformations Although our highest-resolution maps were from particles with the L1 stalk in the closed conformation, we observed additional states of the L1 stalk. We refined several small subsets into low-resolution densities displaying various conformations of L1 and Nmd3 using focused classification with signal subtraction to improve the EM densities by removing particles of alternative conformations (see Appendix Figs S3–S8 for classification strategies, Appendix Table S1 for description of classes). Four primary states were observed for the L1 stalk: closed, open, and two intermediate positions (Fig 4A). Nmd3 density becomes progressively less defined as the L1 stalk moves from the closed to the open state and completely disappears in the open state. The N-terminal domain of Nmd3 can only be observed in maps with L1 in the fully closed state and disappears completely as the L1 stalk transitions toward the open state. In the intermediate positions of the L1 stalk, we were able to rigid body-fit the uL1-Nmd3 structure of the closed complex into the densities. In the different states, the L1 stalk appeared to lift Nmd3 as a single, rigid unit, as both uL1 and Nmd3 maintained the same relative configuration (Fig 4B). However, it is possible that the eL22-like domain of Nmd3 could adopt a different relative conformation in the presence of a P-site ligand. Figure 4. The L1 stalk and Nmd3 adopt multiple states A. Overlap of the four observed states for L1 stalk/Nmd3. T1 (tan) and T2 (purple) are transition states between the fully open (gray) and fully closed (blue). B. Rigid-body docking of uL1-L1-Nmd3 into T2 state, showing the atomic model overlap between the closed (orange) and T2 position (blue). C, D. Model f