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The Ribosomal Stalk Binds to Translation Factors IF2, EF-Tu, EF-G and RF3 Via a Conserved Region of the L12 C-Terminal Domain

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The Ribosomal Stalk Binds to Translation Factors IF2, EF-Tu, EF-G and RF3 Via a Conserved Region of the L12 C-Terminal Domain doi:10.1016/j.jmb.2006.10.025 J. Mol. Biol. (2007) 365, 468–479 The Ribosomal Stalk Binds to Translation Factors IF2, EF-Tu, EF-G and RF3 via a Conserved Region of the L12 C-terminal Domain Magnus Helgstrand1, Chandra S. Mandava2, Frans A. A. Mulder1,3 Anders Liljas3, Suparna Sanyal2⁎ and Mikael Akke1⁎ 1Department of Biophysical Efficient protein synthesis in bacteria requires initiation factor 2 (IF2), Chemistry, Lund University, elongation factors Tu (EF-Tu) and G (EF-G), and release factor 3 (RF3), each Box 124, SE-22100 of which catalyzes a major step of translation in a GTP-dependent fashion. Lund, Sweden Previous reports have suggested that recruitment of factors to the ribosome and subsequent GTP hydrolysis involve the dimeric protein L12, which 2Department of Cell and forms a flexible “stalk” on the ribosome. Using heteronuclear NMR Molecular Biology, Uppsala spectroscopy we demonstrate that L12 binds directly to the factors IF2, University, Box 596, BMC, EF-Tu, EF-G, and RF3 from Escherichia coli, and map the region of L12 SE-75124 Uppsala, Sweden involved in these interactions. Factor-dependent chemical shift changes 3Department of Molecular show that all four factors bind to the same region of the C-terminal domain Biophysics, Lund University, of L12. This region includes three strictly conserved residues, K70, L80, and Box 124, SE-22100 E82, and a set of highly conserved residues, including V66, A67, V68 and Lund, Sweden G79. Upon factor binding, all NMR signals from the C-terminal domain become broadened beyond detection, while those from the N-terminal domain are virtually unaffected, implying that the C-terminal domain binds to the factor, while the N-terminal domain dimer retains its rotational freedom mediated by the flexible hinge between the two domains. Factor- dependent variations in linewidths further reveal that L12 binds to each factor with a dissociation constant in the millimolar range in solution. These results indicate that the L12-factor complexes will be highly populated on the ribosome, because of the high local concentration of ribosome-bound factor with respect to L12. © 2006 Elsevier Ltd. All rights reserved. *Corresponding authors Keywords: L12; ribosome; GTPase; NMR spectroscopy; protein synthesis Introduction In the past few years, structural studies by X-ray crystallography and cryo-electron microscopy (cryo- EM) have revolutionized our understanding of Ribosomes are molecular machines, whose flex- 2–9 ible components enable the dynamic process of ribosome function at atomic resolution. As protein synthesis that includes movement along the increasingly more structural information on the mRNA, tRNA translocation and peptidyl transfer.1 ribosome becomes available, the focus of the field is shifting towards understanding the detailed – motions necessary to accomplish translation.10 14 As a general rule, G-protein translation factors Present address: F. A. A. Mulder, Department of promote the transitions between different structural Biophysical Chemistry, University of Groningen, states of the ribosome, and these processes often Nijenborgh 4, 9747 AG Groningen, the Netherlands. involve GTP hydrolysis in a ribosome-dependent Abbreviations used: CTD, C-terminal domain; EF-G, manner. Protein L12 (also denoted L7/L12) is the elongation factor G; EF-Tu, elongation factor Tu; EM, main component of the prominent “stalk” of the 50 S electron microscopy; HSQC, heteronuclear ribosomal subunit in bacteria, and has been impli- single-quantum correlation; IF2, initiation factor 2; NTD, cated as a key player in factor recruitment and – N-terminal domain; RF3, release factor 3. factor-dependent GTP hydrolysis,15 17 as outlined E-mail addresses of the corresponding authors: further below. Functionally analogous proteins are [email protected]; [email protected] present in eukaryotes and archaea, but these do not 0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. L12 binds to IF2, EF-Tu, EF-G, and RF3 469 show any amino acid sequence homology to the release of inorganic pyrophosphate after GTP – L12.18 20 L12 forms a symmetric dimer mediated hydrolysis.17 A recent cryo-EM reconstruction of EF- by its N-terminal domains (NTD), to which each C- G bound to the ribosome suggests a direct interac- terminal domain (CTD) is connected flexibly via an tion of the CTD of L12 with the G' domain of EF-G.37 – unstructured hinge segment.21 23 High-resolution There is indirect evidence for interactions between structures are available for the isolated CTD of L12 and EF-Tu as well. It has been shown that Escherichia coli L12,24 as well as for the intact E. coli specific mutations in the L12 CTD affect the binding L12 dimer.21 L12 is the only multi-copy protein of of the EF-Tu–GTP–aminoacyl-tRNA ternary com- the ribosome, with two or three dimers binding to plex to the ribosome.38 These data, together with the the ribosome via protein L10, depending on the structural homology between the L12 CTD and EF- organism.16 Recently, the structure of the complex Ts, have been used to generate a model for the between L10 and the dimeric NTDs of L12 from structure of the EF-Tu–L12 complex,38 based on the Thermotoga maritima was determined, which re- crystal structure of the EF-Tu–EF-Ts complex.39 It is vealed that the L12 NTD dimers bind to helix α8 an open question whether L12 also stimulates the of L10. Apparently, the length of α8 determines the GTPase activity of IF2 and RF3. A recent cryo-EM number of L12 dimers that bind to the ribosome in a structure of the initiation complex shows one L12 given organism; helix α8 is longer in Thermotoga L10 CTD intercalated between L11 and the G-domain of and contains one additional (third) binding site for a IF2.9 Because IF2, EF-Tu, EF-G, and RF3 have two dimer of L12, compared with the case in E. coli.16 homologous domains and occupy overlapping sites – Notably, the L12 dimers are highly disordered in all on the ribosome,9,31,32,40 42 it is expected that ribo- high-resolution crystal structures of the 70 S or 50 S some-bound L12 can interact with all of these particles.2,6,7,25 However, from cryo-EM reconstruc- factors. tions of the ribosome in various functional states, it Here, we demonstrate that L12 binds to IF2, EF- appears that the stalk acquires different conforma- Tu, EF-G, and RF3, through a strictly conserved tions during translation. Modeling of the L10– region of its CTD. Estimated affinities between the (L12NTD)4 complex into the electron densities of factors and L12 in solution indicate that the different cryo-EM structures suggests that the corresponding complexes will be highly populated orientation of helix α8 varies between functional on the ribosome. states.16 Furthermore, a body of structural and biochemical data has shown that parts of the hinge region and the entire CTD are mobile in ribosome- Results – bound L12,22,23,26 28 and that its conformation and flexibility are altered dramatically upon binding of Using heteronuclear NMR spectroscopy, we the translation factors EF-G10,23,29,30 or EF-Tu31,32 to investigated the transient interactions between L12 the ribosome. Evidence from chemical cross- and the translation factors IF2, EF-Tu, EF-G, and linking33 and immuno-EM34 studies shows that RF3, as well as bovine serum albumin (BSA), which the CTD contacts different parts of the ribosome, served as a negative control. Separate titration series including the 30 S neck and head regions, as well as were performed for each of IF2, EF-Tu, EF-G, RF3 a position close to the EF-G binding site near L11.35 and BSA, where the unlabeled protein was titrated Thus, there is ample evidence that L12 can attain into a solution of 15N-labeled L12, and a 1H-15N different conformational states during different heteronuclear single-quantum coherence (HSQC) steps of translation. spectrum was acquired at each titration point. In bacteria, translation involves four main G- Interactions between L12 and factor were identified proteins that catalyze different steps. These are: on the basis of changes in chemical shifts and initiation factor 2 (IF2), which is required for linewidths of cross-peaks in the HSQC spectrum of initiation; elongation factor Tu (EF-Tu), which L12, as a function of added factor. The chemical shift carries amino acid-charged tRNAs to the ribosome; is exquisitely sensitive to the magnetic environment elongation factor G (EF-G), which catalyzes tRNA of the nuclear spin, and the linewidth depends on translocation; and release factor 3 (RF3), which the size of the molecular system. Changes in releases the class I release factors after removal of chemical shifts and linewidths provide residue- the nascent peptide.1 All of these factors possess the specific information on changes in the local envir- well-conserved G-domain, which binds and hydro- onment and on the size of the molecular complex, lyzes GTP to carry out their functions, and the because each backbone N-H amide group in L12 succeeding β-barrel domain (domain II). It has been corresponds to a single cross-peak in the 1H-15N shown that L12 alone can stimulate a low level of HSQC spectrum. Thus, these data map out which GTP hydrolysis by EF-G.36 Furthermore, ribosomes parts of L12 interact with each factor. The response depleted of L12 are impaired in the GTPase was very similar in all titration series, and followed activation of both EF-Tu and EF-G.15 The molecular the general features described in detail below for the mechanism of this GTPase activation is not fully interaction between EF-G and L12.
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