The Solution Structure of the Guanine Nucleotide Exchange Domain Of

Home , Eukaryotic translation

View metadata, citation and similar papers at core.ac.uk brought to you by CORE Researchprovided Articleby Elsevier -217 Publisher Connector

The solution structure of the guanine nucleotide exchange domain of human elongation factor 1b reveals a striking resemblance to that of EF-Ts from Escherichia coli Janice MJ Pérez1,2‡, Gregg Siegal2*‡, Jan Kriek1, Karl Hård2†, Jan Dijk1, Gerard W Canters2 and Wim Möller1

Background: In eukaryotic protein synthesis, the multi-subunit elongation Addresses: 1Department of Molecular Cell Biology, factor 1 (EF-1) plays an important role in ensuring the fidelity and regulating the Sylvius Laboratory, University of Leiden, Wassenaarseweg 72, NL-2333 AL Leiden, The rate of translation. EF-1α, which transports the aminoacyl tRNA to the Netherlands and 2Leiden Institute of Chemistry, β ribosome, is a member of the G-protein superfamily. EF-1 regulates the activity Gorlaeus Laboratory, University of Leiden, of EF-1α by catalyzing the exchange of GDP for GTP and thereby regenerating Einsteinweg 55, NL-2333 CC Leiden, The the active form of EF-1α. The structure of the bacterial analog of EF-1α, EF-Tu Netherlands. has been solved in complex with its GDP exchange factor, EF-Ts. These †Present address: Astra Structural Chemistry structures indicate a mechanism for GDP–GTP exchange in prokaryotes. Laboratory, S-43183 Mölndal, Sweden. Although there is good sequence conservation between EF-1α and EF-Tu, there is essentially no sequence similarity between EF-1β and EF-Ts. We ‡These two authors contributed equally to this work. wished to explore whether the prokaryotic exchange mechanism could shed any *Corresponding author. light on the mechanism of eukaryotic translation elongation. E-mail: [email protected]

Results: Here, we report the structure of the guanine-nucleotide exchange Key words: G protein, guanine-nucleotide factor (GEF) domain of human EF-1β (hEF-1β, residues 135–224); exchange factor, NMR, translation elongation β hEF-1 [135–224], determined by nuclear magnetic resonance spectroscopy. Received: 8 October 1998 Sequence conservation analysis of the GEF domains of EF-1 subunits β and δ Revisions requested: 19 November 1998 from widely divergent organisms indicates that the most highly conserved Revisions received: 4 December 1998 residues are in two loop regions. Intriguingly, hEF-1β[135–224] shares Accepted: 14 December 1998 structural homology with the GEF domain of EF-Ts despite their different Published: 1 February 1999 primary sequences. Structure February 1999, 7:217–226 Conclusions: On the basis of both the structural homology between EF-Ts http://biomednet.com/elecref/0969212600700217 and hEF-1β[135–224] and the sequence conservation analysis, we © Elsevier Science Ltd ISSN 0969-2126 propose that the mechanism of guanine-nucleotide exchange in protein synthesis has been conserved in prokaryotes and eukaryotes. In particular, Tyr181 of hEF-1β[135–224] appears to be analogous to Phe81 of Escherichia coli EF-Ts.

Introduction The intrinsic dissociation rate of GDP from EF-1α is slow In eukaryotes, the pentameric elongation factor 1 (EF- (~0.7 × 10–3/sec), resulting in the accumulation of inactive α βγδ α 1 2 ) ensures rapid transport and accurate delivery of EF-1 that would lead to the eventual stoppage of transla- aminoacyl tRNA (aa-tRNA) to the ribosome [1]. The tion [4]. The dissociation rate of GDP from EF-1α is EF-1α subunit is recruited to the endoplasmic reticulum enhanced 3000-fold by the catalytic action of a complex of by interaction with the EF-1βγδ complex, which is the β and γ subunits of EF-1 [4]. The EF-1βγ complex pro- embedded in the membrane [2]. GTP binding to EF-1α vides a target for the regulation of the overall rate of transla- produces a conformation that is active for binding of tion within the cell. The goal of the present work was to aa-tRNA and thus allows the ternary complex of EF-1α– further understand the nature of this regulation through GTP–aa-tRNA to deliver the aa-tRNA to the A (acceptor) analysis of the GDP–GTP exchange mechanism at the site of the ribosome [3] (Figure 1). Correct codon–anti- molecular level. codon interaction is required to induce hydrolysis of GTP to GDP, which results in a conformational change in In addition to its role in protein synthesis, EF-1α has been EF-1α that causes release of both the aa-tRNA and the reported to function in a number of other cellular processes ribosome. The ribosome then catalyzes peptide-bond for- including organization of the cytoskeleton [5], cell division mation between the incoming aa-tRNA and the peptidyl [6], cell proliferation [7], ageing [8,9] and regulation of the site (P site) bound nascent chain in the elongation phase rate of apoptosis [10]. Recently, it has been shown that the of protein synthesis. entire EF-1 complex is an essential component of the 218 Structure 1999, Vol 7 No 2

Figure 1 G proteins share a common structural domain, the so- called ‘G domain’, that is responsible for binding guanine nucleotides [15]. The architecture of the domain includes GDP GTP EF-1αβ six β strands surrounded by five to six α helices [16]. Two EF-1α–GDP regions called switch I and switch II contribute to guanine- EF-1α–GTP nucleotide binding. The conformation of these regions is EF-1β aa-tRNA γ 5′ 3′ sensitive to the presence of the phosphate of the bound mRNA E P A nucleotide and therefore provides the basis for the regula- EF-1α–GTP–aa-tRNA tion of activity. The structure of the bacterial homologue of EF-1α, EF-Tu, has been solved in complex with GDP 5′ 3′ A E mRNA P [17], a GTP analog (Gpp(NH)p) [18,19] and its GEF, Pi EF-Ts [20,21]. The prokaryotic and eukaryotic proteins consist of three domains. Domain I, the G domain, binds α EF-1 –GTP–aa-tRNA the guanine nucleotide, whereas domains II and III are ribosome Structure responsible for aa-tRNA binding. The molecular basis Schematic diagram of the role of elongation factor 1 (EF-1) in for the GDP-exchange mechanism in bacteria has been translation. At the top, the free complex of the α and β subunits ascribed to the insertion of the sidechain of EF-Ts Phe81 (EF-1αβ) is shown. Binding of GTP to this complex results in the (sPhe81) between helices B and C of EF-Tu and the β α displacement of EF-1 and the activation of EF-1 for aa-tRNA consequent disruption of the Mg2+–GDP binding site binding. The ternary complex can then bind to a ribosome (schematically shown as the 40S and 60S subunits with tRNA [gray [20,21]. (Following the nomenclature in [20], amino acid cloverleaf] occupying the peptidyl [P] and exit [E] sites), in which residues from EF-Ts and EF-Tu are prefixed by an ‘s’ the acceptor (A) site is free, forming the quaternary complex or a ‘u’, respectively). In particular, the conformational depicted at the bottom. Upon correct codon–anti-codon interaction, changes brought about by EF-Ts binding to EF-Tu the GTP is hydrolyzed to GDP and the aa-tRNA is released to the ribosome (schematically shown with all three sites occupied and the result in the ‘flipping’ of the peptide bond of the highly incoming amino acid as a black circle). The inactive EF-1α–GDP conserved uVal20. Consequently, the CO oxygen of complex is a target for binding of EF-1β, which then catalyzes the uVal20 and the β phosphate of GDP are brought into release of GDP, allowing the reactivation of EF-1α through passive close juxtaposition, causing electrostatic clash and eject- binding of GTP. ing GDP. This has been termed the ‘phosphate-side-first’ mechanism [22]. EF-1β shares a high degree of sequence homology with EF-Tu, especially in the guanine nucleo- RNA polymerase of the vesicular stomatitis virus [11], a tide binding regions. This sequence conservation has situation reminiscent of the Qβ RNA phage replicase, allowed the construction of a homology-based model of the which associates with the bacterial elongation factor. three-dimensional (3D) structure of EF-1α [23]. However, However, the exact role of EF-1α in this diverse array of there is essentially no sequence similarity between EF-1β, events is still unclear. EF-1δ and EF-Ts. The combined lack of structural and biochemical data makes it impossible to reliably predict EF-1α is a member of the G-protein family, as it uses the mechanism of GDP–GTP exchange catalyzed by GTP hydrolysis as a switching mechanism to regulate its EF-1β or EF-1δ. activity [12,13]. In fact, although it was not recognized as such at the time, EF-1α is one of the archetypal members We have shown that the GEF activity resides in the highly of this large and important family of proteins that includes, conserved C-terminal domain of both EF-1β and EF-1δ as well as the initiation, elongation and release factors in [24]. As EF-1β forms a stable complex with EF-1α in vivo protein synthesis, Ras and Ras-like proteins, the so-called and in vitro and appears to be the minimal GEF required ‘small G proteins’, and the heterotrimeric G proteins to support active translation, our work has focused on this involved in signal transduction [13]. G proteins are func- subunit. We have recombinantly expressed a number of tionally active when in the GTP-bound state and inactive proteins of different lengths comprising the C-terminal in the GDP-bound state. Down-regulation of G proteins portion of EF-1β, all of which proved to be active in can occur through enhancement of the GTPase activity guanine nucleotide exchange assays and formed com- by so-called GAPs (GTPase activating proteins). In the plexes with EF-1α [25]. Gel filtration indicates that these case of EF-1α, no soluble GAP activity has been found. complexes contain a 1:1 stoichiometry of EF-1α:EF-1β Conversely, G proteins can be activated via the catalyti- [25]. The smallest polypeptide, hEF-1β[135–224], which cally induced exchange of bound GDP for GTP by GEFs was 91 amino acid residues long, proved to be the most (guanine-nucleotide exchange factors). Two different GEF stable and possessed the most favorable NMR characteris- proteins for EF-1α have been found, namely EF-1β and tics (numbering of amino acid residues refers to the intact, EF-1δ [14], which have a high level of sequence similarity wild-type protein) [25]. In order to gain a better under- and form part of the EF-1 complex in vivo. standing of the mechanism of GDP–GTP exchange in Research Article GEF domain of EF-1b Pérez et al. 219

protein synthesis, we have isotopically labeled and com- of the NOE distance restraints versus the residue number pleted the resonance assignment of hEF-1β[135–224] is graphically depicted in Figure 2. No NOE distance using double- and triple-resonance NMR techniques [26]. restraints were found for iMet, Leu135, Tyr181 and Here, we report the solution structure of the GEF domain Gly182. Final structures were calculated from 50 initial of EF-1β. Remarkably, hEF-1β[135–224] shows a striking structures with randomized coordinates using the simu- resemblance to the structure of its bacterial counterpart, lated-annealing protocol of DYANA performed in dihe- EF-Ts. On the basis of this comparison, a molecular dral-angle space. The 20 structures with the lowest target mechanism has been proposed for GDP–GTP exchange functions were used to represent the solution conforma- in eukaryotic cells. tion of hEF-1β[135–224].

Results and discussion NMR analysis and structure calculation of hEF-1b[135–224] Figure 2 The solution structure of the guanine nucleotide exchange domain from human EF-1β (hEF-1β[135–224]) has been β1 α1 β 2 β 3 α 2 β 4 determined by heteronuclear, multi-dimensional NMR spectroscopy. Assignment of backbone and sidechain res- (a) onances was accomplished by a combination of double- and triple-resonance experiments previously described 60 [26]. The resonances of the HN protons of each of the first two N-terminal residues, initiator Met (iMet) and Leu135, as well as Tyr181 and Gly182, could not be 40 detected in NMR experiments employing presaturation for solvent suppression. Use of pulsed-field gradients in conjunction with water flip-back techniques to avoid Number of NOEs solvent saturation resulted in the observation of a weak 20 signal from the amide proton of Gly182, while those of residues iMet, Leu135 and Tyr181 remained unde- tectable. This fact implies that the amide proton of these residues is in rapid chemical exchange with protons of (b) the solvent. The crosspeak of Gly180 is also weaker than average in HN proton-detected experiments. In agree- ment with the experimental data, Gly180, Tyr181 and 6.0 Gly182 form a loop between β strands 2 and 3 in the calculated structures, which would be expected to show reduced amide-proton protection with respect to residues 4.0 in regular secondary structure. Displacement/Å The final structure calculation was based on 1412 distance restraints (429 intra-residue, 323 sequential, 201 medium- 2.0 range and 459 long-range) and 70 backbone dihedral-angle restraints (Table 1). Distance restraints were obtained from both a 3D [15N,1H] nuclear Overhauser effect spec- 143 153 163 173 183 193 203 213 troscopy heteronuclear single quantum coherence (NOESY- Residue number Structure HSQC) [27] and a simultaneous acquisition 3D [15N/13C,1H]- 3 NOESY-HSQC experiment [28]. Backbone JHNα coupling constants were derived from a heteronuclear multiple Plots of the number of NOE restraints and rms displacement versus. residue number for hEF-1β[135–224]. (a) Distribution of NOE-based quantum coherence (HMQC-J) experiment on uniformly distance restraints versus primary sequence for hEF-1β[135–224]. 15N-labeled protein [29]. Crosspeaks in the NOESY spectra The elements of secondary structure, as determined from the derived from short 1H–1H distances in regular secondary ensemble of 20 structures, are schematically indicated on top of the β α structure or unambiguously assignable from chemical- figure (arrows for strands and cylinders for helices). The number of significant NOE restraints (as determined by the program DYANA) is shift information alone were assigned manually. This shown on the left: open bars, intraresidual; light shading, sequential; resulted in some 700 distance restraints. The resonances dark shading, medium range; black, long range). (b) Plot of the global of the remaining NOE crosspeaks were assigned in an auto- residue displacement [56] versus primary sequence for mated manner using the NOAH routine in the program hEF-1β[135–224]. The displacement of backbone atoms amongst the 20 structures of the ensemble is shown by the solid line, whereas the DYANA version 1.4 [30,31]. The assignments made in displacement for all heavy atoms is denoted by the dotted line. NOAH were then cross-checked manually. The distribution 220 Structure 1999, Vol 7 No 2

The 20 final structures had a total of nine NOE Table 1 violations > 0.2 Å out of 28,200 input restraints. No viola- Summary of structure statistics. tion exceeded 0.38 Å. There were no dihedral angle > 〈 〉 〈 〉 restraint violations 2° (Table 1). The small size and SA * SAr * number of residual constraint violations shows that the input data represent a self-consistent set and that the Number of input restraints Intraresidual 429 restraints are well satisfied in the calculated conformers Sequential 323 (Table 1). As a further check, structures were calculated Medium range 201 using an identical set of distance restraints as input, but Long range 459 with dihedral-angle restraints derived from the Cα Backbone-angle restraints 70 chemical shift (as implemented in DYANA v.1.4) instead Violations of upper distance limits Number > 0.2 Å 9 0 3 of from JHNα coupling constants. No additional distance Rmsd (Å) 0.018 0.019 restraints were violated, nor were the original angle Maximum (Å) 0.38 0.17 restraints that were derived from the coupling constants. Violations of torsion-angle restraints Number > 2° 0 0 Rmsd (°) 0.004 0.004 The Ramachandran map generated in PROCHECK Maximum (°) 0.09 0.04 NMR [32] indicates that the majority of the residues Coordinate precision (Å) (85%) have angular averages in energetically favorable residues 138–178 and 183–223† ± regions, 14% in additional allowed regions and 1.4% in the Backbone-atom rmsd 0.57 0.08 Heavy-atom rmsd 1.11 ± 0.13 generously allowed regions. DYANA target function (Å2) 0.69 ± 0.1 1.08

Structure description of hEF-1b[135–224] *〈SA〉 represents the ensemble of 20 structures calculated by simulated annealing in dihedral-angle space in DYANA1.4. 〈SA 〉 represents the A stereo diagram of the backbone trace of a superposition r energy-minimized mean structure calculated as follows. The Cα atoms of of the 20 structures with the lowest target function is residues 5–45 and 50–90 of the ensemble of structures were shown in Figure 3. The root mean square deviation superimposed using MOLMOL [54]. Further alignment was achieved (rmsd) for the backbone heavy atoms of residues 138–178 using the ‘flip’ command and subsequently the mean structure was and 183–223 is 0.57 (± 0.08) Å and 1.11 (± 0.13) Å for all determined by averaging of the coordinates of all atoms. The mean structure was then subjected to 1000 steps of restrained energy heavy atoms of these residues. Overall, the structure is minimization in cartesian space using X-PLOR 3.851 [55]. Finally, this well defined apart from the four N-terminal amino acids structure was read back into DYANA1.4, where it was subjected to and the loop between strands β2 and β3 (residues 4200 steps of restrained conjugate gradient minimization. †Coordinate 180–182). This can be seen clearly in Figure 2b, where precision is defined as the rmsd from each of the 20 members of the ensemble to the energy-minimized mean structure based on backbone the rms displacement versus sequence number is graphi- superposition of residues 5–45 and 50–90. cally depicted. Relatively few NOE interactions could be found for residues in the β2β3 loop and the structure is therefore poorly defined by the data. This loop is most are strongly protected from the solvent. A few prominent probably undergoing chemical exchange, as evidenced by features of the structure are worth mentioning. First, only the lack of protection of the amide protons. The one of the six aromatic residues in EF-1β (Trp171) is truly sequence Gly–Tyr–Gly would be expected to be intrinsi- buried within the hydrophobic core of the protein (14% cally flexible because of a lack of steric constraint on the solvent-exposed surface area). The other five lie along the backbone and thus may also be experiencing conforma- surface and vary from having 35% solvent-exposed surface tional exchange. The lack of detectable sidechain reso- (Trp148) to 70% (Tyr181). Second, 85% of the final struc- nances of Tyr181 supports this hypothesis. tures include an H bond between Hε1 of Trp148 and the backbone CO of Glu210. This polar interaction appears to The structure is a two layered α/β sandwich composed of lock the loop between helix α2 and strand β4 into a highly two anti-parallel α helices overlaying a four-stranded anti- rigid conformation, as can be seen by the low displace- parallel β sheet (Figure 4). The β sheet is formed by ment value in Figure 2b. Third, in 12 of the 20 conform- residues 138–147 (β1), 170–178 (β2), 183–189 (β3) and ers, the sidechain carboxylate of Asp200 provides an 213–223 (β4). The last strand (β4) is irregular and forms a N-terminal cap for helix α2. β bulge from residues 220 to 222. These residues show the enhanced NH-exchange that is characteristic of a β bulge. As a first approach to determining the functionally impor- The α helices are formed by residues 155–163 (α1) and tant residues of hEF-1β[135–224], we quantitatively ana- 199–207 (α2). A hydrophobic core is formed by residues lyzed the residue-by-residue sequence conservation. A Ile141, Leu142, Leu143, Val145, Pro147, Thr152, Met154, BLAST search was performed on all nucleic acid and Leu157, Val161, Ile164, Leu169, Trp171, Leu186, Ile188, protein databases using the entire 91-residue sequence of Cys190, Val192, Val197, Leu202, Ile206 and Val213. These EF-1β. After removal of redundancies and non-related residues are primarily hydrophobic and their amide protons proteins, 25 sequences remained of EF-1β and EF-1δ pro- Research Article GEF domain of EF-1b Pérez et al. 221

Figure 3

Stereoview of the backbone (N, Cα, C′) of the best-fit superposition of the final 20 selected conformers of hEF-1β[135–224]. Every tenth Cα atom has been labeled. The strands of the β sheet are shown in red, the α helices are in green and the loops are in blue. This figure was prepared using the program MOLMOL [54].

teins from archaebacteria, plants, fungi and animals. The L7/L12 [40]. Cousineau and colleagues [41] have pro- sequences were aligned using the program PileUp and posed a model to explain the conservation of this fold, were analyzed using Plotsim in the GCG package. The which involves a series of gene duplication/fusion events matrix correlation of Plotsim was modified such that sub- that would have taken place before the divergence of the stitution of Phe for Tyr resulted in no penalty, whereas three superkingdoms: eubacteria, archaea, and eukaryotes. substitution of Ala for Gly was only penalized 15%. The In their model, the root of this protein family would have results are depicted graphically in Figure 4. As expected, a been a small RNA-binding domain of the α/β type. number of residues that contribute to the hydrophobic core or form a tight turn, such as Val145, Gly168, Val170, Identification of a possible GDP–GTP exchange Trp171, Gly172 and Gln214, are moderately well con- mechanism served. In addition, residues in the C-terminal half of The crystal structure of bacterial EF-Tu has been solved strand β4, which includes the β bulge, are shown to be in complex with its GEF protein, EF-Ts from two differ- weekly conserved. However, the two regions having the ent species [20,21]. In both structures there are only a greatest sequence conservation are the loops between β1 small number of interactions between the GEF domain of and α1 (Pro147–Trp148–Asp149–Asp150) and between EF-Ts (residues 57–139 in E. coli) and the G domain of β2 and β3 (Gly180–Tyr181–Gly182). Conservation of EF-Tu that are important for GDP release. As shown in residues in loops is not as common as for residues in Figure 5a, all residues of EF-Tu that participate in these regular secondary structure and points to a functional, interactions with EF-Ts are conserved in EF-1α. In con- rather than structural, evolutionary constraint. trast, there is very little sequence homology between EF-1β and EF-Ts (Figure 5b). Furthermore, the sequen- The secondary structure elements are sequentially con- tial pattern of secondary structure elements is completely nected in the order β1α1β2β3α2β4, and form an αβ sand- different in the two proteins, thus rendering global wich superfold (Figure 4) [33]. This fold, with the same sequence alignment meaningless (Figure 5c). In spite of βαββαβ motif, is found in a number of functionally unre- this difference, the 3D architecture of the GEF domain of lated and nonhomologous proteins, for example, ferre- EF-Ts is surprisingly similar to the solution structure of doxin [34], MerP [35], Menkes ATPase [36], the RNP hEF-1β[135–224] (Figure 6a). Superposition of EF-1β domain of U1AsnRNP [37], the prokaryotic translation- and the GEF domain of EF-Ts results in an rmsd of initiation factor IF3 [38] and the prokaryotic ribosomal 0.76 Å for 19 Cα atoms in three β strands and two α helices protein S6 [39]. A more distant relationship, in the sense (Figure 6b). In this orientation, helix α1 of hEF-1β− that the fold is conserved despite the insertion of an extra [135–224] superimposes well with helix h5 of EF-Ts, while helix, is found in the prokaryotic ribosomal protein α2 lies close to helix h6 (nomenclature of Kawashima et al. 222 Structure 1999, Vol 7 No 2

Figure 4 expect this sequence conservation from observation of the structure of hEF-1β[135–224], as the Tyr181 sidechain is on a mobile loop and is 70% solvent-exposed. The combination of the structural similarity between hEF-1β[135–224] and EF-Ts, and the highly conserved nature of Tyr181, suggest that it functions analogously to sPhe81. This suggestion is supported by the fact that the residues that form the hydrophobic pocket on EF-Tu into which sPhe81 is inserted (sHis84, sHis118 and sLeu121) are conserved in EF-1α (Figure 5a). Riis et al. have devel- oped a model for human EF-1α based upon homology to the G-protein family [23]. In this model, His95, His136 and Leu139 have the same orientation as their bacterial homologs and form a similar hydrophobic pocket. There- fore it is likely that Tyr181 of EF-1β inserts between helices B and C of EF-1α, thus disrupting the Mg2+-binding site and causing the release of GDP.

The residue sPhe81 lies within a sequence motif, QTDFV (single-letter amino acid code; consensus sequence ETDFV) that is highly conserved amongst prokaryotic and eukaryotic mitochondrial EF-Ts proteins. A similar sequence, ETDM, has been found to be conserved amongst the eukaryotic EF-1β and EF-1δ subunits. On the basis of this sequence similarity, Kawashima and colleagues suggested that this motif may play a similar role to the ETDFV motif in EF-Ts [20]. However, in our structure the ETDM sequence (residues 151–154) forms a well-defined loop in which the Met154 Cα is approximately 11 Å from the Cα of sPhe81. Additionally, the Ribbon diagram of the mean structure of hEF-1β[135–224]. The sidechain of Met154 is buried (only 16% solvent-exposed secondary structural elements are labeled as defined in the text. The surface area) and forms part of the hydrophobic core of ribbon has been colored to reflect the quantitative level of sequence β conservation as described in the Materials and methods section. All hEF-1 [135–224]. Therefore, major structural rearrange- residues below the mean level of conservation are colored blue. ment would be required in order for the sidechain of Residues between the mean and the maximum are colored on a linear Met154 to interact with EF-1α. scale from blue to yellow, such that red is precisely halfway between the mean and the maximum. This figure was prepared using the program MOLMOL [54]. Analysis of the crystal structure of the EF-Ts–EF-Tu complex suggests that key interprotein contacts are conserved in the analogous eukaryotic complex. The superposition of EF-Ts and EF-1β in Figure 6b indicates that [20]). In the complex with EF-Tu, the GEF domain of three residues of EF-Ts that make important contacts EF-Ts contacts the G domain in an end-on manner with with EF-Tu have functional analogs in EF-1β. First, the respect to the axes of helices h5 and h6 and the β sheet. helix capping function of sAsp80, which serves to reori- The half of the β sheet that lies closest to EF-Tu in the ent EF-Tu helix B, may be mimicked by the backbone complex then superposes well with three of the four CO of Pro178 of EF-1β. Second, the hydrogen bond β β strands in hEF-1 [135–224] (Figure 6b). between the sidechain NH2 of uGln114 and the backbone CO of sIle125, could be conserved through the Tyr181 of hEF-1β[135–224] is located in a loop, the con- interaction of the sidechain carboxylate of EF-1β Asp150 formation of which is poorly defined by the NMR data, that (in the β1α1 loop) with Gln132 of EF-1α (Figure 5a). may exhibit conformational flexibility. In the bundle of This hydrogen bond serves to stabilize the reorientation 20 structures (Figure 3), the sidechain of Tyr181 occupies of EF-Tu helix C. Third, the hydrogen bond acceptor one of two positions. In half of the structures, superposition function of the CO oxygen of sGly126 could be mimic- in the above manner places Tyr181 of hEF-1β[135–224] ked by the sidechain carboxylate of EF-1β Asp149 (also and sPhe81 in approximately the same position in space in the β1α1 loop), thus conserving the crucial hydrogen (Figure 6b). As demonstrated in Figure 4, Tyr181 is highly bond interaction with Hε1 of uHis19 (His15 of EF-1α, conserved in EF-1β and EF-1δ proteins. One would not Figure 5a). This interaction is particularly important as it Research Article GEF domain of EF-1b Pérez et al. 223

Figure 5

Sequence and secondary structure comparison of human and bacterial elongation (a) P Loop factor subunits. (a) Sequence alignment of Switch I ecEF-Tu 1 SKEKFERTKPHVNVGTIGHVDHGKTTLTAAITTVLAKT------YGGAARA the GTP binding domains of E. coli EF-Tu hEF-1α 1 ---MGKE-KTHINIVVIGHVDSGKSTTTGHLIYKCGGIDKRTIEKFEKEAAEMGKGSFKY (ecEF-Tu) and human EF-1α (hEF-1α). Strand A Helix A Identical residues are indicated by a vertical Switch I Switch II line between the sequences, whereas similar ecEF-Tu 46 FDQIDNAPEEKARGITINTSHVEYDTPTRHYAHVDCPGHADYVKNMITGAAQMDGAILVV residues are indicated by a dot. The hEF-1α 57 AWVLDKLKAERERGITIDISLWKFETSKYYVTIIDAPGHRDFIKNMITGTSQADCAVLIV secondary structure is labeled according to Strand B Strand CHelix B Strand D convention and indicated by arrows (β strands) and cylinders (α helices). All ecEF-Tu 106 AATDGPM------PQTREHILLGRQVGVPYIIVFLNKCDMVDD------EELLELVEM residues that contact the bound nucleotide in hEF-1α 117 AAGVGEFEAGISKNGQTREHALLAYTLGVKQLIVGVNKMDSTEPPYSQKRYEEIVKEVST the structure of ecEF-Tu are boxed and Helix C Strand E Helix D labeled with the common nomenclature, as α are the homologous residues in EF-1 . (b) 144 154164 174 184 β1 α 1 β2 β3 Residues important for GDP release are hEF-1β 135 ....LVAKSSILLDVKPWDDETDMAKLEECVRSIQADGLVWGSSKLVPVGYGIKKLQIQC indicated by black vertical arrows and ecEF-Ts 62 KTK.IDGNYGIILEVNCQTDFVAKDAGFQAFADKVLDAAVAG..KTDVEVLKAQFEEERV residues important for intersubunit contact are s1 s2 h4 h5 h6 194α 2 204 214 β4 indicated by open vertical arrows. hEF-1β 191 VVEDDKVGTDMLEEQ..ITA.FEDYVQSMDVAAFNKI (b) Sequence comparison of ecEF-Ts 120 ALVAKIGENINIRRVAALEG hEF-1β[135–224] and the GEF domain of h7 s3 E. coli EF-Ts (ecEF-Ts; residues 62–139). Secondary structure elements are indicated (c) as described above. The residues of EF-Ts helix 4, which are primarily responsible for the h4 GEF reaction, are boxed. The proposed h7 structurally equivalent residues in EF-1β are s3s1 s2 h5 α 2 β 4 β 1 β 3 β 2 α 1 also boxed. (c) Schematic diagram of the h6 sequential connectivity of the secondary structural elements of hEF-1β[135–224] and the GEF domain of E. coli EF-Ts (ecEF-Ts). Helices are indicated by cylinders and the ecEF-Ts EF-1β strands of the β sheet are indicated by arrows. The nomenclature of ecEF-Ts is that Structure used in [17].

would stabilize the ‘flipped’ conformation about EF-1α [43,44], RCC1 (regulator of chromosome condensation) Val16, which would result in electrostatic clash between [45] and Sos (cell proliferation and differentiation) [46]. the Val16 CO oxygen and the β phosphate of GDP. Despite the structural similarity of the G proteins they These last two postulated interactions would explain the interact with, the structure of each of these GEFs is very extremely strong sequence conservation in the loop different and bears no resemblance to hEF-1β[135–224] or between strand β1 and helix α1 in EF-1β and EF-1δ pro- EF-Ts. Thus far, the mechanism of GDP release by the teins from different species (Figure 4). GEF for each of the three classes of G protein seems to differ. The first, as exemplified by the elongation factors, Initial attempts at characterizing the EF-1α–EF-1β complex involves insertion of residues in a helical loop of the GEF by NMR were not successful because of protein aggrega- between the Switch II and helix C region of the G protein tion. However, even if this were not the case, it would [20,21]. It is possible that this method, or a variant of it, still not be possible to differentiate contacts between is also used by Mss4 [42] and RCC1 [45], both of which hEF-1β[135–224] and the G domain of EF-1α or domains contain loops shown to interact with their G proteins. II and III. Therefore, confirmation of our hypothesis must Mossessova et al. suggest that this mechanism may also be await the results of mutagenesis studies and NMR experi- mimicked by the ARNO-Sec7–Arf interaction [43]. In the ments on the complex of hEF-1β[135–224] and the iso- second group, a helical hairpin of the GEF protein Sos lated G domain of EF-1α. is inserted into Ras, causing a large movement of the switch I region and a major structural change in the Comparison with known GEF structures switch II region [46]. The third group is represented by The past few years have seen the publication of a number the heterotrimeric G proteins and their activation by hep- of structures of eukaryotic GEF proteins that are involved tahelical transmembrane receptors. Conserved regions of in different cellular processes, including Mss4 (vesicular contact between the α and β subunits lie in a hydrophobic transport) [42], ARNO-Sec7 (secretory vesicle coating) pocket formed by the switch II region of the α subunit 224 Structure 1999, Vol 7 No 2

Figure 6

Comparison of the tertiary structure of EF-1β and the GEF domain of EF- and yellow, β sheet in dark blue) is shown on the right, with the loop Ts. (a) Side-by-side ribbon diagrams of the GEF domain of E. coli EF-Ts between β2 and β3 in magenta and the sidechain of Tyr181 in yellow. (residues 57–139) and hEF-1β[135–224]. The structure on the left is (b) Superposition of the GEF domain of EF-Ts and hEF-1β[135–224]. the GEF-domain of EF-Ts (α helices in red and yellow, β sheet in cyan) The color scheme is identical to that in (a). Nineteen Cα atoms from from the complex with EF-Tu [20]. The sPhe81 sidechain is shown in each structure were chosen for a least-squares superposition. This figure dark blue. The mean structure of hEF-1β[135–224] (α helices in green was prepared using the program MOLMOL [54].

[47]. It is proposed that the β subunit opens the nucleo- prokaryotes and eukaryotes. This strengthens the idea tide-binding site by displacing switch I and that receptor that the wealth of structural information available on binding induces a conformational change in a loop that bacterial elongation factors is applicable to eukaryotes. contacts the guanine ring [48]. A surprising result of these Despite the low sequence similarity, the fold of studies is the incredible variation about some unifying hEF-1β[135–224] determined here bears a striking resem- principles which are aimed at the goal of releasing GDP blance to that of the GEF domain of bacterial EF-Ts, without blocking the ability of GTP to bind. particularly those regions that interact with EF-1α. This may therefore represent a case of convergent evolution. Biological implications On the basis of both structural similarity and the pattern Elongation factor 1 (EF-1) plays a pivotal role in the main- of amino acid sequence conservation amongst EF-1β and tenance and regulation of protein biosynthesis. EF-1α in EF-1δ subunits, we propose a mechanism for GDP–GTP its active conformation binds and transports amino- exchange in eukaryotic cells in which Tyr181 of EF-1β acyl tRNA (aa-tRNA) to the ribosome. The β subunit, plays an analogous role to Phe81 of E. coli EF-Ts. EF-1β, is responsible for the regeneration of the active form of EF-1α. Regeneration is achieved through catal- The presence of recognition sites for a number of protein ysis of the exchange of GDP for GTP on EF-1α, which kinases on all four subunits of EF-1 (α,β,γ and δ) indicates provides for regulation of the elongation stage of transla- the importance of exchange factors in the cellular regulation. In eukaryotes, little is known of the molecular tion of protein synthesis [49]. Knowledge of the mecha- mechanism by which GDP is ejected from EF-1α, nism of GDP–GTP exchange will help to guide further thereby allowing binding of GTP. As a first step in under- studies to determine how this regulation is effected and standing this reaction, we have solved the solution struc- how it may go wrong in various disease states. ture of the guanine-nucleotide exchange factor (GEF) domain (residues 135–225) of human EF-1β. With the recent elucidation of the structures of a number of GEFs, the variety of possible mechanisms to effect The present study suggests that, on the whole, the three- this reaction is becoming clear. The observation that dimensional structure of both the aa-tRNA binding and the bacterial mechanism of GDP–GTP exchange is the GEF subunits of EF-1 has been conserved in conserved in the eukaryotic elongation factors and that Research Article GEF domain of EF-1b Pérez et al. 225

the key residues for GEF activity are present in two the structures was checked using the programs ACQUA and solvent-exposed loops suggests that Mss4 and RCC1 PROCHECK-NMR [32]. may utilize a similar mechanism. Furthermore, it is pos- Sequence-conservation analysis sible that the three proposed methods of GEF function BLAST (performed at NCBI using the BLAST network service now cover the entire repertoire that exists in nature. http://www.ncbi.nlm.nih.gov/BLAST/) was used to search all available nucleic acid and protein databases with the 91-residue sequence of β β The phenomenon of 3D structure conservation in spite hEF-1 [135–224]. In all, 25 sequences were found comprising EF-1 and EF-1δ proteins from a wide array of species in archaebacterial, of different primary sequences has been observed previ- fungal, plant and animal kingdoms. The 25 sequences were aligned ously. Interestingly, the three-dimensional fold of two using the program PILEUP and the residue-by-residue conservation α helices packed against an antiparallel β sheet is found was quantitatively analysed in the program PLOTSIM in the GCG in many RNA-binding proteins. This suggests that this package (Wisconsin Package, v.9.1, Genetics Computer Group, (GCG) Madison, Wisconsin, USA). The 2D matrix of residue similarity small domain could be at the origin of many of the pro- was modified such that replacement of Tyr with Phe resulted in the teins involved in translation. same score as no replacement. The score of a Gly to Ala replacement was set to 85% of the conservation score of absolute conservation. Materials and methods Accession numbers NMR sample preparation β U-15N, 13C labeled hEF-1β[135–224] was expressed in E. coli, purified The coordinates for hEF-1 [135–224] have been deposited with the and tested for GDP–GTP exchange activity as described previously [26]. Brookhaven Protein Databank under accession number 1b64. The NMR samples contained 1 mM hEF-1β[135–224] in 10 mM sodium Acknowledgements phosphate pH 6.9, 1 mM DTT, 100 mM sodium chloride, 0.02% NaN3 β We thank Cees A van Sluis for help with the sequence-conservation analysis. and 5% D2O. Iodoacetylation of the two cysteines in native hEF-1 indicates that they are reduced. We therefore kept the recombinant protein under reducing conditions for NMR studies. References 1. Janssen, G.M., van Damme, H.T., Kriek, J., Amons, R. & Möller, W. NMR spectroscopy (1994). The subunit structure of elongation factor 1 from Artemia. Why two alpha-chains in this complex? J. Biol. Chem. 269, 31410- All NMR experiments were performed at 30°C using a Bruker DMX-600 31417. spectrometer equipped with a pulsed-field gradient accessory and a 2. Sanders, J., Brandsma, M., Janssen, G.M., Dijk, J. & Möller, W. 5 mm triple-resonance probe. The program NMRPipe [50] was used for (1996). Immunofluorescence studies of human fibroblasts transformation of the time domain data. NMR spectra were analyzed and demonstrate the presence of the complex of elongation factor-1 peak-integrated using the program XEASY [51]. NOE-based distance beta gamma delta in the endoplasmic reticulum. J. Cell. Sci. 109, restraints were obtained from both a 3D [15N, 1H]-edited NOESY-HSQC 1113-1117. spectrum (τ = 150 ms, 768(t3) × 48(t2) × 96(t1) complex points) [27] 3. Negrutskii, B.S. & El’skaya, A.V. (1998). Eukaryotic translation and a simultaneous acquisition 3D [15N/13C, 1H]-edited NOESY-HSQC elongation factor-1alpha: structure, expression, functions and possible role in amino acyl-tRNA channeling. Prog. Nucleic Acid Res. and Mol. spectrum (τ = 100 ms, 1024(t3) × 32(t2) × 128(t1) complex points) [28]. 60 3 Biol. , 47-77. JHNHα coupling constants were derived from an HMQC-J experiment 4. Janssen, G.M. & Möller, W. (1988). Kinetic studies on the role of [29]. No coupling constants could be determined for residues iMet, elongation factors 1 beta and 1 gamma in protein synthesis. J Biol. Leu135, Pro147, Pro178, Tyr181 and Gly182. Chem. 263, 1773-1778. 5. Liu, G., Edmonds, B.T. & Condeelis, J. (1996). pH, EF-1α and the Determination of the 3D structure cytoskeleton. Trends Cell. Biol. 6, 168-171. Initially, only NOESY crosspeaks whose identity was unambiguous, 6. Kuryama, R., Savereide, P., Lefebvre, P. & Dasgupta, S. (1990). The on the basis of chemical-shift data alone, were assigned. This predicted amino acid sequence of a centrosphere protein in dividing sea urchin eggs is similar to elongation factor (EF-1α). J. Cell Sci. 95, resulted in approximately 700 distance restraints. The complete list 3 231-236. with all peaks integrated and a list of JHNHα coupling constants was 7. Tatsuka, M., Mitsui, H., Wada, M., Nagata, A., Nojima, H. & Okayama, used as input for the NOAH routine within the DYANA 1.4 package H. (1992). Elongation factor-1α gene determines susceptibility to [30,31]. NOAH uses structures calculated from the input data as a transformation. Nature 359, 333-336. constraint to further assign NOE peaks that are ambiguous with 8. Stearns, S.C. & Kaiser, M. (1993). The effects of enhanced expression ∆ α respect to chemical-shift data. The tol value for the NOAH calcula- of elongation factor EF-1 on lifespan in Drosophila melanogaster. tion was 0.02 ppm for protons and 0.1 ppm for heavy atoms. NOAH Genetica 91, 167-182. itself was run with default parameters, which resulted in the further 9. Shikama, N., Ackerman, R. & Brack, C. (1994). Protein synthesis α assignment of approximately 700 crosspeaks. All isolated NOE dis- elongation factor EF-1 expression and longevity in Drosophila 91 tance restraints (as defined by a low score using the ‘distance check’ melanogaster. Proc. Natl Acad. Sci. USA , 4199-4203. 10. Duttaroy, A., Bourbeau, D., Wang, X. & Wang, E. (1998). Apoptosis command in DYANA) were inspected manually for correctness. rate can be accelerated or decelerated by overexpression or reduction of the level of Elongation Factor-1α. Exp. Cell Res. 238, 168-176. The peak lists derived from the NOAH calculation were converted 11. Das, T., Mathur, M., Gupta, A.K., Janssen, G.M.C. & Banerjee, A.K. into distance restraints using the program CALIBA [52]. The peak (1998). RNA polymerase of vesicular stomatitis virus specifically calibration was based upon known distances in regular secondary associates with translation elongation factor-1 for its activity. Proc. 3 Natl Acad. Sci. USA 95,1449-1454. structure [53]. Dihedral-angle restraints were calculated from JHNHα coupling constants using the program HABAS [52]. This program 12. Möller, W. & Amons, R. (1985). Phosphate-binding sequences in also provided χ1 angle restraints for residues containing two β-meth- nucleotide-binding proteins. FEBS Lett. 186, 1-7. 13. Bourne, H.R., Sanders, D.A. & McCormick, F. (1990). The GTPase ylene protons. No stereospecific assignments or hydrogen-bond con- superfamily: a conserved switch for diverse cell functions. Nature 348, straints were included in the final structure calculation. Fifty 125-132. structures were calculated from random starting coordinates using 14. van Damme, H.T.F., Amons, R., Karssies, R., Timmers, C.J., Janssen, simulated annealing in dihedral-angle space as implemented in G.M.C. & Möller, W. (1990). Elongation factor 1β of Artemia: DYANA. The 20 conformers with the lowest target function were localization of functional sites and homology to elongation factor 1δ. accepted as representative of the solution structure. The quality of Biochim. Biophys. Acta 1050, 241-247. 226 Structure 1999, Vol 7 No 2

15. Bourne, H.R., Sanders, D.A. & McCormick, F. (1991). The GTPase 41. Cousineau, B., Leclerc, F. & Cedergreen, R. (1997). On the origin of superfamily: conserved structure and molecular mechanism. Nature protein synthesis factors: a gene duplication/fusion model. J. Mol. 349, 117-127. Evol. 45, 661-670. 16. Hilgenfeld, R. (1995). Regulatory GTPases. Curr. Opin. Struct. Biol. 42. Yu, H. & Schreiber, S.L. (1995). Structure of guanine-nucleotide- 5, 810-817. exchange factor human Mss4 and identification of its Rab-interacting 17. Kjeldgaard, M. & Nyborg, J. (1992). Refined structure of Elongation surface. Nature 376, 788-791. Factor EF-Tu from Escherichia coli. J. Mol. Biol. 223, 721-742. 43. Mossessova, E.M., Gulbis, J.M. & Goldberg, J. (1998). Structure of the 18. Berchtold, H., Reshetnikova, L., Reiser, C.O.A., Schirmer, N.K. Sprinzl, guanine nucleotide exchange factor Sec7 domain of human Arno and M. & Hilgenfeld, R. (1993). Crystal structure of active Elongation analysis of the interaction with ARF GTPase. Cell 92, 415-423. Factor Tu reveals major domain rearrangements. Nature 365, 44. Cherfils, J., et al., & Chardin, P. (1998). Structure of the Sec7 domain 126-132. of the Arf exchange factor ARNO. Nature 392, 101-105. 19. Kjeldgaard, M., Nissen, P., Thirup, S. & Nyborg, J. (1993). The crystal 45. Renault, L., et al., & Wittinghofer, A. (1998). The 1.7 Å crystal structure of elongation factor EF-Tu from Thermus aquaticus in the structure of the regulator of chromosome condensation (RCC1) GTP conformation. Structure 15, 35-50. reveals a seven-bladed propeller. Nature 392, 97-101. 20. Kawashima, T., Berthet-Colominas, C., Wulff, M., Cusack, S. & 46. Boriack-Sjodin, P.A., Margarit, S.M., Bar-Sagi, D. & Kuriyan, J. (1998). Leberman, R. (1996). The structure of the Escherichia coli EF-Tu·EF- The structural basis of the activation of Ras by Sos. Nature 394, Ts complex at 2.5 Å resolution. Nature 379, 511-518. 337-343. 21. Wang, Y., Jiang, Y., Meyering-Voss, M., Sprinzl, M. & Sigler, P.B. 47. Lambright, D.G., Sondek, J., Bohm, A., Skiba, N.P., Hamm, H.E. & (1997). Crystal structure of the EF-Tu·EF-Ts complex from Thermus Sigler, P.B. (1996). Nature 379, 311-319. thermophilus. Nat. Struct. Biol. 4, 650-656. 48. Iiri, T., Farfel, Z. & Bourne, H.R. (1998). Nature 394, 35-38. 22. Bohm, A., Gaudet, R. & Sigler, P.B. (1997). Structural aspects of 49. Janssen, G.M., Maessen, G.D., Amons, R. & Möller, W. (1988) heterotrimeric G-protein signaling. Curr. Opin. Biotech. 8, 480-487. Phosphorylation of elongation factor 1 beta by an endogenous kinase 23. Riis, B., Rattan, S.I.S., Clark, B.F.C. & Merrick, W.C. (1990). Eukaryotic affects its catalytic nucleotide exchange activity. J. Biol. Chem. 263, protein elongation factors. Trends Biochem. Sci. 15, 420-424. 11063-11066. 24. van Damme, H.T.F., Amons, R., Janssen, G.M.C. & Möller, W. (1991). 50. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. & Bax, A. Mapping the functional domains of the eukaryotic elongation factor (1995). NMRPipe: a multidimensional spectral processing system 1βγ. Eur. J. Biochem. 197, 505-511. based on UNIX pipes. J. Biomol. NMR 6, 277-293. 25. Pérez, J.M.J., Kriek, J., Dijk, J., Canters, G.W. & Möller W. (1998). 51. Bartels, C., Xia, T., Billeter, M., Güntert, P. & Wüthrich, K. (1995). The Expression, purification and spectroscopic studies of the guanine program XEASY for computer-supported NMR spectral analysis of nucleotide exchange domain of human elongation factor, EF-1β. biological macromolecules. J. Biomol. NMR 5, 1-10. Protein Express. Purif. 13, 259-267. 52. Güntert, P., Braun, W. & Wüthrich, K. (1991). Efficient computation of 26. Pérez, J.M.J., et al., & Canters, G.W. 1H, 15N and 13C chemical shift three-dimensional protein structures in solution from nuclear magnetic assignment of the guanine nucleotide exchange domain of human resonance data using the program DIANA and the supporting programs Elongation Factor-one beta. J. Biomol. NMR 12, 467-468. CALIBA, HABAS and GLOMSA. J. Mol. Biol. 217, 517-530. 27. Cavanaugh, J., Fairbrother, W.G., Palmer III, A.G. & Skelton, N.J. 53. Wüthrich, K. (1986). NMR of Proteins and Nucleic Acids. John Wiley (1996). Heteronuclear-edited NMR spectroscopy. In Protein NMR & Sons, Inc., New York. Spectroscopy: Principles and Practice, pp. 447-468, Academic 54. Koradi, R., Billeter, M. & Wüthrich, K. (1996). MOLMOL: a program for Press, San Diego, USA. display and analysis of macromolecular structures. J. Mol. Graph. 14, 28. Pascal, S.M., Muhandiram, D.R., Yamazaki, T., Forman-Kay, J.D. & Kay, 51-55. L.E. (1994). Simultaneous acquisition of 15N- and 13C-edited NOE 55. Brünger, A.T. (1992). X-PLOR Version 3.1. A System for X-ray spectra of proteins dissolved in H2O. J. Magn. Reson. B 103, 197-201. Crystallography and NMR. Yale University Press, New Haven, CT, USA. 29. Kay, L.E. & Bax, A. (1990). New methods for the measurement of NH- 56. Billeter, M., Kline, A.D., Braun, W., Huber, R & Wüthrich, K. (1989). CαH coupling constants in 15N-labeled proteins. J. Magn. Reson. 86, Comparison of the high-resolution structures of the α-amylase 110-126. inhibitor Tendamistat determined by nuclear magnetic resonance in 30. Güntert, P., Mumenthaler, C. & Wüthrich, K. (1997). Torsion angle solution and by X-ray diffraction in single crystals. J. Mol. Biol. 206, dynamics for NMR structure calculation with the new program 677-687. DYANA. J. Mol. Biol. 273, 283-298. 31. Mumenthaler, C., Güntert, P., Braun, W. & Wüthrich, K. (1997). Automated combined assignment of NOESY spectra and three- dimensional protein structure determination. J. Biomol. NMR 10, 351-362. 32. Laskowski, R.A., Rullmann, J.A.C., MacArthur, M.W., Kaptein, R. & Thornton, J.M. (1996). AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477-486. 33. Orengo, C.A. & Thornton, J.M. (1993). Alpha plus beta folds revisited: some favoured motifs. Structure 1, 105-120. 34. Adman, E.T., Sieker, L.C., & Jensen, L.H. (1976). Structure of Peptococcus aerogenes ferredoxin. J. Biol. Chem. 251, 3801-3806. 35. Steele, R.A. & Opella, S.J. (1997). Structures of the reduced and mercury-bound forms of MerP, the periplasmic protein from the bacterial mercury detoxification system. Biochemistry 36, 6885-6895. 36. Gitschier, J., Moffat, B., Reilly, D., Wood, W.I. & Fairbrother, W.J. (1998). Solution structure of the fourth metal-binding domain from the Menkes copper-transporting ATPase. Nat. Struct. Biol. 5, 47-54. 37. Nagai, K., Oubridge, C., Jessen, T.H., Li, J. & Evans, P.R. (1990). Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A. Nature 348, 515-520. 38. Garcia, C., Fortier, P., Blanquet, S., Lallemand, J. & Dardel, F. (1995). Solution structure of the ribosome-binding domain of E. coli Translation Initiation Factor IF3. Homology with the U1A protein of the Because Structure with Folding & Design operates a eukaryotic spliceosome. J. Mol. Biol. 254, 247-259. 39. Lindahl, M. & Liljas, A. (1994). Crystal structure of the ribosomal ‘Continuous Publication System’ for Research Papers, this protein S6 from Thermus thermophilus. EMBO J. 13, 1249-1254. paper has been published on the internet before being printed 40. Leijonmarck, M. & Liljas, A. (1987). Structure of the C-terminal domain of the ribosomal protein L7/L12 from Escherichia coli at 1.7 Å. J. Mol. (accessed from http://biomednet.com/cbiology/str). For Biol. 195, 555-580. further information, see the explanation on the contents page.