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Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 A resolution

2000; Springer Nature; Volume: 19; Issue: 11 Linguagem: Inglês

10.1093/emboj/19.11.2710

1460-2075

Marı́a G. Gómez-Lorenzo, Christian M. T. Spahn, Rajendra K. Agrawal, Robert A. Grassucci, Pawel A. Penczek, Kalpana Chakraburtty, Juan P. G. Ballesta, José Luís Lavandera, José Garcia-Bustos, Joachim Frank,

Genomics and Phylogenetic Studies

Article1 June 2000free access Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 Å resolution Maria G. Gomez-Lorenzo Maria G. Gomez-Lorenzo Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Research Department, Glaxo Wellcome, S.A., Severo Ochoa 2, 28760 Tres Cantos, CSIC and UAM de Madrid, Canto Blanco, 28049 Madrid, Spain, USA Search for more papers by this author Christian M.T. Spahn Christian M.T. Spahn Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Howard Hughes Medical Institute, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Rajendra K. Agrawal Rajendra K. Agrawal Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Robert A. Grassucci Robert A. Grassucci Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Howard Hughes Medical Institute, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Pawel Penczek Pawel Penczek Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Kalpana Chakraburtty Kalpana Chakraburtty Medical College of Wisconsin, Department of Biochemistry, Milwaukee, WI, 53226 USA Search for more papers by this author Juan P.G. Ballesta Juan P.G. Ballesta Centro de Biología Molecular 'severo Ochoa', CSIC and UAM de Madrid, Canto Blanco, 28049 Madrid, Spain Search for more papers by this author Jose L. Lavandera Jose L. Lavandera Research Department, Glaxo Wellcome, S.A., Severo Ochoa 2, 28760 Tres Cantos, CSIC and UAM de Madrid, Canto Blanco, 28049 Madrid, Spain, USA Search for more papers by this author Jose F. Garcia-Bustos Jose F. Garcia-Bustos Research Department, Glaxo Wellcome, S.A., Severo Ochoa 2, 28760 Tres Cantos, CSIC and UAM de Madrid, Canto Blanco, 28049 Madrid, Spain, USA Search for more papers by this author Joachim Frank Corresponding Author Joachim Frank Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Howard Hughes Medical Institute, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Howard Hughes Medical Institute, Wadsworth Center, POB 509, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Maria G. Gomez-Lorenzo Maria G. Gomez-Lorenzo Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Research Department, Glaxo Wellcome, S.A., Severo Ochoa 2, 28760 Tres Cantos, CSIC and UAM de Madrid, Canto Blanco, 28049 Madrid, Spain, USA Search for more papers by this author Christian M.T. Spahn Christian M.T. Spahn Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Howard Hughes Medical Institute, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Rajendra K. Agrawal Rajendra K. Agrawal Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Robert A. Grassucci Robert A. Grassucci Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Howard Hughes Medical Institute, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Pawel Penczek Pawel Penczek Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Kalpana Chakraburtty Kalpana Chakraburtty Medical College of Wisconsin, Department of Biochemistry, Milwaukee, WI, 53226 USA Search for more papers by this author Juan P.G. Ballesta Juan P.G. Ballesta Centro de Biología Molecular 'severo Ochoa', CSIC and UAM de Madrid, Canto Blanco, 28049 Madrid, Spain Search for more papers by this author Jose L. Lavandera Jose L. Lavandera Research Department, Glaxo Wellcome, S.A., Severo Ochoa 2, 28760 Tres Cantos, CSIC and UAM de Madrid, Canto Blanco, 28049 Madrid, Spain, USA Search for more papers by this author Jose F. Garcia-Bustos Jose F. Garcia-Bustos Research Department, Glaxo Wellcome, S.A., Severo Ochoa 2, 28760 Tres Cantos, CSIC and UAM de Madrid, Canto Blanco, 28049 Madrid, Spain, USA Search for more papers by this author Joachim Frank Corresponding Author Joachim Frank Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Howard Hughes Medical Institute, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA Howard Hughes Medical Institute, Wadsworth Center, POB 509, Empire State Plaza, Albany, NY, 12201-0509 USA Search for more papers by this author Author Information Maria G. Gomez-Lorenzo1,2, Christian M.T. Spahn1,3, Rajendra K. Agrawal1,4, Robert A. Grassucci1,3, Pawel Penczek1,4, Kalpana Chakraburtty5, Juan P.G. Ballesta6, Jose L. Lavandera2, Jose F. Garcia-Bustos2 and Joachim Frank 1,3,4,7 1Health Research Inc. at Wadsworth Center, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA 2Research Department, Glaxo Wellcome, S.A., Severo Ochoa 2, 28760 Tres Cantos, CSIC and UAM de Madrid, Canto Blanco, 28049 Madrid, Spain, USA 3Howard Hughes Medical Institute, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA 4Department of Biomedical Sciences, State University of New York at Albany, Empire State Plaza, Albany, NY, 12201-0509 USA 5Medical College of Wisconsin, Department of Biochemistry, Milwaukee, WI, 53226 USA 6Centro de Biología Molecular 'severo Ochoa', CSIC and UAM de Madrid, Canto Blanco, 28049 Madrid, Spain 7Howard Hughes Medical Institute, Wadsworth Center, POB 509, Empire State Plaza, Albany, NY, 12201-0509 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2710-2718https://doi.org/10.1093/emboj/19.11.2710 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome·EF2 complex at 17.5 Å resolution by three-dimensional (3D) cryo-electron microscopy. EF2 is directly visible in the 3D map and the overall arrangement of the complex from Saccharomyces cerevisiae corresponds to that previously seen in Escherichia coli. However, pronounced differences were found in two prominent regions. First, in the yeast system the interaction between the elongation factor and the stalk region of the large subunit is much more extensive. Secondly, domain IV of EF2 contains additional mass that appears to interact with the head of the 40S subunit and the region of the main bridge of the 60S subunit. The shape and position of domain IV of EF2 suggest that it might interact directly with P-site-bound tRNA. Introduction The elongation cycle of protein synthesis consists of three basic steps: A-site occupation, peptidyl transfer and translocation. While the peptidyl transferase activity is an intrinsic activity of the large ribosomal subunit, the rates of A-site occupation and translocation are greatly enhanced by soluble elongation factors. EF-Tu in prokaryotes and the homologous EF1α in eukaryotes form a ternary complex with the aminoacylated tRNA (aa-tRNA) and GTP that delivers the aa-tRNA into the ribosomal A-site. The translocation reaction is mediated by EF-G in prokaryotes and the homologous EF2 in eukaryotes. All these factors belong to the G-protein superfamily (Bourne et al., 1991). For a long time, it was generally accepted that these elongation factors follow the classical scheme of G-proteins. They successively bind to the ribosome in the GTP conformation, catalyze their respective step on the ribosome, subsequently hydrolyze GTP and dissociate in the GDP conformation. More recently, this view has been challenged in the case of the translocation reaction and it was suggested that EF-G and possibly EF2 may act like force-generating motor proteins (Abel and Jurnak, 1996; Rodnina et al., 1997). Because of the high degree of evolutionary conservation of the protein synthesis machinery (rRNA, ribosomal proteins, translation factors), the general framework of protein synthesis is believed to be the same in prokaryotes and eukaryotes. However, eukaryotic 80S ribosomes are considerably larger than the prokaryotic 70S ribosomes and also protein synthesis appears to be more complex in eukaryotes (see Hershey et al., 1996). Apart from translation initiation, which is far more complicated in eukaryotes, there are also significant differences in the ribosomal elongation phase. In addition to the GTP molecules hydrolyzed by EF-Tu/EF1α and EF-G/EF2, eukaryotes consume ATP during the elongation cycle. In fungi this ATP hydrolysis takes place on a third elongation factor, EF3 (Skogerson and Wakatama, 1976). Polypeptide chain elongation in fungi requires EF3-dependent ATP hydrolysis to release deacylated tRNA from the ribosomal E-site (Triana-Alonso et al., 1995). Cryo-electron microscopy (cryo-EM) has been used in recent years to study the structure of the ribosome and its binding to functional ligands. Even at moderate resolutions, in the range 15–20 Å, binding positions and conformational changes of elongation factors could be deduced from the resulting density maps (Stark et al., 1997; Agrawal et al., 1998, 1999). It has recently been found that the interaction of EF2 with the ribosome is the cellular target for members of the sordarin family of antifungal compounds (Capa et al., 1998; Dominguez and Martin, 1998; Dominguez et al., 1998; Gomez-Lorenzo and Garcia-Bustos, 1998; Justice et al., 1998, 1999). Even though there is a high degree of homology between the fungal and mammalian protein synthesis machineries, these inhibitors are highly specific for the fungal elongation step (Kinsman et al., 1998). This study was prompted by the possibility that sordarin compounds could stabilize the yeast ribosome·EF2 complex and allow its imaging by cryo-EM, as has been previously accomplished with fusidic acid and the Escherichia coli ribosome·EF-G complex (Agrawal et al., 1998). Sordarin derivative GM193663 was used to stabilize the ribosome·EF2 complex and to obtain the first three-dimensional (3D) reconstruction of a eukaryotic 80S ribosome in complex with one of several protein factors of the protein synthesis machinery. The resolution of the map, 17.5 Å, is the highest obtained so far for a eukaryotic ribosome. It has allowed us to determine the binding position of EF2 and to discuss the similarities and possible differences in the mechanism of translocation between prokaryotes and eukaryotes. Results Overall structure of the yeast 80S ribosome Using micrographs at several defoci, contrast transfer function (CTF) correction, and a much larger dataset, we were able to drastically improve the resolution of the cryo-EM map of the yeast 80S ribosome from Saccharomyces cerevisiae. Previous reconstructions of the yeast 80S ribosome yielded resolutions of 35 Å for the empty ribosome (Verschoor et al., 1998) and of 26 Å for an 80S–Sec61 complex (Beckmann et al., 1997). Here we present the structure of the S.cerevisiae 80S ribosome in complex with elongation factor EF2. The complex was stabilized by using the sordarin derivative GM193663, a translational inhibitor that is specific for fungi and probably acts in a way similar, although not identical, to fusidic acid in prokaryotes (Capa et al., 1998; Dominguez et al., 1998, 1999; Justice et al., 1998; Kinsman et al., 1998). Approximately 60% of the 80S ribosomes carried EF2 (see Table I). This is lower than the corresponding binding of EF-G to the E.coli 70S ribosomes (80%), but compares very well with the reported 60% activity of yeast ribosomes in tRNA binding (Triana-Alonso et al., 1995). Table 1. EF2 and sordarin binding to 80S ribosomes Background 80S EF2 80S + EF2 Sordarin (pmol)v 0.2 0.2 0.9 2.3 v – – – 0.58 In a total volume of 50 μl, 5 pmol of 80S ribosomes and 51 pmol of EF2 were incubated in the presence of [3H]sordarin (20 μM, f.c.). Controls were performed using only sordarin, 80S ribosomes and sordarin, or EF2 and sordarin. After the binding reaction (see Materials and methods) the sample was passed through a PD10 gel filtration column. The amount of sordarin present in the flow-through was determined by measuring the radioactivity in the flow-through. The occupancy v (pmol sordarin/pmol 80S ribosomes) was calculated taking the value of EF2 + sordarin as the background value and taking into account that only 2.4 pmol of 80S were present in the flow-through. The resolution curve for the final reconstruction is shown in Figure 1. With the conservative cut-off value of 0.5 (see Malhotra et al., 1998 for discussion), the resolution achieved is 17.5 Å, but the reconstruction still contains information of lower accuracy beyond this point (13.4 Å with the 3-σ criterion; see Orlova and van Heel, 1997). A control reconstruction of the empty ribosome yielded a resolution of 18.9 Å (see Figure 1), confirming that, among other factors, the stabilization of the conformation by binding of the ligand is an important factor in the improvement of resolution. Figure 1.Fourier shell correlation curve, indicating a resolution of 17.5 Å with a 0.5 cut-off criterion for the 80S·EF2·sordarin complex (solid line) and 18.9 Å for the vacant 80S ribosome (dashed line). Download figure Download PowerPoint Since the resolution is in the same range as for published 3D cryo-EM reconstructions of the E.coli 70S ribosome (Agrawal et al., 1998, 1999), a direct comparison of both structures is possible (Figure 2). The similarity between the two structures observed at lower resolution (Verschoor et al., 1998) has become more pronounced. Landmarks observed in the large subunit (central protuberance, stalk, L1 protuberance, tunnel through the subunit) and the small subunit (head, body, platform, shoulder) all bear a detailed resemblance. However, the yeast ribosome has additional elements distributed over the periphery of the structure, which are due to the expansion segments in the rRNA (Dube et al., 1998b; Spahn et al., 1999) and 24 additional proteins (Warner, 1999). Another difference is in the relative arrangement of the subunits (Figure 2). This difference is more complicated than a mere rotation of the 40S subunit, found by Dube et al. (1998a) for the mammalian ribosome. To analyze the changes, we aligned several landmark features of the large subunits of the E.coli 70S and the S.cerevisiae 80S ribosome, i.e. the central protuberance, the L7/L12 stalk, the tunnel and the main bridge B2 (see Frank et al., 1995a for a definition). This analysis shows that when compared with the 70S ribosome, the shoulder of the small subunit in the 80S ribosome is shifted away from the large subunit, while its head is located closer to the central protuberance and its platform is closer to the large subunit. Furthermore, one striking difference is observed in the arrangement of components within the 60S subunit: the L1 protuberance is shifted towards the central protuberance of the 60S subunit when compared with its position in the 50S subunit (Figure 2G–I). Figure 2.Reconstructions of the 80S ribosome from S.cerevisiae without (A, D, G) and with (B, E, H) EF2 bound, and the EF-G-bound 70S ribosome from E.coli (C, F, I; adapted from Agrawal et al., 1999), presented in three equivalent views. Upper row (A–C): side view, with small subunit on the left and large subunit on the right, showing the binding sites of the elongation factors; middle row (D–F): view from the small subunit solvent side, with (E) and (F) showing the extended stalk (St); bottom row (G–I): view from the L1 protein side. The inset above (G) and (H) shows a comparison of the L1 region [within the dashed boundary in (G) and (H)] with the 'split' appearance of L1 in the yeast ribosome in a previous reconstruction (Beckmann et al., 1997). EF2 and EF-G are shown in red. Small insets on the left depict the 80S control map with 40S and 60S colored in yellow and blue, respectively, in corresponding orientations as an interpretation aid. Landmarks, small subunit: h, head; b, body; pt, platform; sh, shoulder (for the designation of subunit body, see Figure 3; large subunit: CP, central protuberance; L1, L1 protuberance; St, extended stalk. Download figure Download PowerPoint Localization and interaction of EF2 with the ribosome An analysis of the difference map between the 80S·EF2·sordarin complex and the vacant ribosome gives a distinct density corresponding to EF2 (see the supplementary data at The EMBO Journal Online) and reveals a large conformational shift of the stalk region upon EF2 binding, with EF2 located close to the base of the stalk and the A-site (see below). The EF2 occupancy of ribosomes was at least 60%, sufficient to observe EF2 directly in the 3D map (Figure 2B and E). The fraction of vacant 80S ribosomes present in the preparation ( 80% for E.coli). However, it can be clearly seen that the stalk extends outwards from the ribosome towards the cytosol, in agreement with its proposed role as an early anchoring point for soluble factors (van Agthoven et al., 1977; Möller and Maassen, 1986; Moazed et al., 1988; Uchiumi et al., 1990). In addition, a second conformational change can be observed in the stalk region: the whole stalk region moves towards the central protuberance (Figure 5). This movement is indicated by a comparison of the positive and negative masses in the difference map between the 80S·EF2 complex and the vacant 80S ribosome, and can also be observed directly (Figure 5). Such a drastic movement in response to EF2 binding has not been observed in the bacterial complex (Agrawal et al., 1998, 1999). Figure 5.Top view of the 80S ribosome (A) without and (B) with EF2 bound, indicating the structural rearrangement of the stalk region. The outline of the stalk region in the 80S·EF2·sordarin complex is indicated by the white dashed line. Small inset on the left: control map (A) with 40S and 60S colored in yellow and blue, respectively, as an interpretation aid. Download figure Download PowerPoint Discussion The reconstruction of the sordarin-stabilized 80S·EF2 complex represents the highest resolution density map of a eukaryotic ribosome obtained so far. We believe that the improvement from 26 Å (Beckmann et al., 1997) to 17.5 Å (Figure 1) is due to three main factors: (i) conformational homogeneity and stability; (ii) increased cryo-EM dataset; and (iii) the use of multiple defocus groups. In the case of the fMet-tRNAfMet·ribosome complex of E.coli (Malhotra et al., 1998), higher resolution could be obtained than with the vacant ribosome, and it was conjectured that as a rule, functionally meaningful complexes might be better defined conformationally than their vacant counterparts. The large jump in resolution in the current study seems to be due, at least in part, to the stabilizing effect of EF2 binding, in agreement with the earlier observations. There is no doubt that another reason for the improvement in resolution is the increase in the number of particles, now at 17 716, especially as it is combined with the strategy of collecting images over a wide range of defoci and CTF correction. As a result, the density map is well defined, with high-resolution features such as thin intersubunit bridges observed for the E.coli ribosome (Frank et al., 1995a, b), and without a noticeable prevalence of feature