The Crystal Structure of the Carboxy-Terminal Domain of Human Translation Initiation Factor eIF5

J. Mol. Biol. (2006) 360, 457–465

doi:10.1016/j.jmb.2006.05.021

The Crystal Structure of the Carboxy-Terminal Domain of Human Translation Initiation Factor eIF5 Christoph Bieniossek 1 †, Patrick Schütz 1 †, Mario Bumann 1 Andreas Limacher 1,2 , Isabel Uson 3 and Ulrich Baumann 1 ⁎ 1

Departement für Chemie und Biochemie, Universität Bern, Freiestrasse 3, CH-3012 Bern, Switzerland 2

Institut für Pharmazeutische Wissenschaften, Swiss Federal Institute of Technology Zürich (ETH), Hoenggerberg, HCI Building, CH-8093 Zurich, Switzerland 3

Institut de Biologia Molecular de Barcelona, Jordi Girona 18–26, 08034 Barcelona, Spain *Corresponding author

The carboxy-terminal domain (CTD) of eukaryotic initiation factor 5 (eIF5) plays a central role in the formation of the multifactor complex (MFC), an important intermediate for the 43 S preinitiation complex assembly. The IF5-CTD interacts directly with the translation initiation factors eIF1, eIF2β, and eIF3c, thus forming together with eIF2 bound Met-tRNAiMet the MFC. In this work we present the high resolution crystal structure of eIF5CTD. This domain of the protein is exclusively composed out of alphahelices and is homologous to the carboxy-terminal domain of eIF2B-ε (eIF2Bε-CTD). The most striking difference in the two structures is an additional carboxy-terminal helix in eIF5. The binding sites of eIF2-β, eIF3 and eIF1 were mapped onto the structure. eIF2-β and eIF3 bind to nonoverlapping patches of negative and positive electrostatic potential, respectively. © 2006 Elsevier Ltd. All rights reserved.

Keywords: translation initiation; HEAT domain; eIF5; multi-factor complex

Introduction Translation initiation is the rate-limiting step during polypeptide synthesis and hence an important point of regulation. In eukaryotes there are three different mechanisms of translation initiation, with the Cap-dependent initiation being the most frequent and best understood of those (for reviews see1,2). At least 11 eukaryotic translation initiation factors (eIFs) are involved in this process. A crucial step is the recognition of the start codon during the scanning process of the 43S preinitiation complex. This complex consists of the small ribosomal subunit bound to the eIF2-GTP-Met-tRNAiMet ternary complex (TC) and translation initiation factors eIF1, eIF1A, eIF3 and eIF5, all of them bound to the TC.3 Base-pairing of the Met-tRNAi with the proper AUG codon triggers GTP hydrolysis by the γ-subunit of eIF2. This GTPase activity is induced by the N-terminal † C.B. and P.S. contributed equally to the work. Abbreviations used: eIF, eukaryotic translation initiation factor; CTD, carboxy-terminal domain; MFC, multifactor complex; PDB, Protein Data Bank; RMS, root-mean-square. E-mail address of the corresponding author: [email protected]

domain of eIF5 which acts as a GTPase activating protein (GAP), probably by inserting an ‘‘arginine finger’’ (Arg-15) into the active site of eIF2-γ.4 GTP hydrolysis evokes the release of eIF2-GDP and presumably of other bound translation initiation factors and therefore prepares for the joining of the large subunit. The carboxy-terminal domain (CTD) of eIF5 was shown to serve as a core of the ribosomal preinitiation complex formation,3,5,6 a function which is apparently independent of its GTPase-activating function. eIF5-CTD binds with distinct interfaces to eIF1, the c-subunit of eIF3, and to eIF4G as well as to the beta subunit of eIF2, the GTPase-activating protein substrate. eIF5-CTD binding to those factors together with the eIF2 bound Met-RNAiMet builds the so-called multifactor complex (MFC).3,7 There is some evidence that the MFC binds as a preformed unit to the 40S ribosomal subunit.8,9 Moreover, mutations relaxing the stringency of start codon selection have been mapped in eIF1, eIF5 and all three subunits of eIF2 and proposed that the integrity of the MFC on the ribosome is a prerequisite for the function of these factors at the AUG selection step.5 Thus, the primary function of eIF5CTD is to serve as an assembly guide by rapidly promoting stoichiometric MFC assembly while excluding formation of non-functional complexes.10

0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.

Crystal Structure of eIF5-CTD

458 In addition, eIF5-CTD binding to eIF4G is thought to mediate at least partially the binding of the mRNA Cap-eIF4F complex to the 43S complex. The minimal domains for eIF5-CTD binding have been mapped within eIF2-β, eIF3c and eIF4G. In eIF2-β the amino-terminal half with its three lysinerich segments (K-boxes) mediates a tight eIF5-CTD binding, whereas K-box 2 was shown to be the essential unit of interaction.3,11,12 Furthermore, an amino-terminal serine-rich acidic segment of eIF3c and an expanded HEAT domain of eIF4G are responsible for eIF5-CTD binding.3,13 For eIF5 the binding motif is located at its very carboxyterminus and is formed by a bipartite region dominated by aromatic and acidic amino acids called AA-boxes or W2 domain (two invariant tryptophans, 11,14,15 Trp354 and Trp381 in Figure 1). Such AA-boxes can also be found in the carboxy-termini of all eukaryotic eIF2B-ε proteins (the catalytic subunit of eIF2B) and in mammalian eIF4G. The crystal structure of the AA-box comprising domain of eIF2B-ε, almost equivalent in length to eIF5-CTD, was solved and revealed an all helical protein with AA-box 1 being located at its surface and AA-box 2 being not visible in the electron-density-map.16 The eight helices are organized in a manner similar to HEAT repeats with the AA-boxes 1 and 2 being

located between helix VI and VII, and after helix VIII, respectively. The structure displays a highly asymmetric molecule with respect to surface charge and conservation. Here we present the structure of the carboxyterminal domain of human eIF5 (eIF5-CTD). The solved structure reveals a high similarity to eIF2BεCTD. The most striking difference between the two molecules is an additional carboxy-terminal helix in the new structure. This helix makes it possible to predict the course of the charged AA-box 2 which could not be observed in the previous structure of eIF2Bε-CTD. This second AA-box runs almost perpendicular to AA-box 1 and seems to play a major role in the interaction towards other translation initiation factors such as eIF2.

Results Binding assay The eIF5-CTD construct described here binds to eIF2-β(1–192) (human eIF2-β comprising amino acids 1 to 192) in a salt dependent manner as demonstrated by gelfiltration experiments. Figure 2 shows the elution profile and SDS-PAGE analysis of

Figure 1. Multiple sequence alignment of eIF5 C-terminal domains. Shown are the C-terminal sequences of human (UniProt P55010), mouse (P59325), Schizosaccharomyces pombe (Q76N36), Saccharomyces cerevisiae (P38431), and Arabidopsis thaliana (Q9XI91) eIF5 proteins. Secondary structure elements are indicated according to the human eIF5-CTD X-ray structure presented here. The figure was prepared with ESPRIPT.34

Crystal Structure of eIF5-CTD

459

Figure 2. Gelfiltration binding assay. (a) Depicted is the gelfiltration peak profile and corresponding SDS gel fractions of the eIF5-CTD-eIF2-β(1–192) complex preincubated and run with low salt (buffer B1). (b) Shows the same experiment using a buffer containing 100 mM NaCl (buffer B2). The complex is clearly dissociated with the two proteins eluting as separate peaks. Experiments at 4C and room temperature were performed and yielded identical results. The volumes of elution (in [ml]) are indicated on top of each peak with the corresponding relative molecular weight (deduced from experiments with proteins standards). The bands of the SDS gels are marked with the corresponding protein names.

the fractions. The experiment clearly indicates an eIF5-CTD-eIF2-β(1–192) complex formation under low salt conditions (20 mM NaCl and less). Running the experiment with the same buffer containing more than 50 mM NaCl leads to a dissociation of the two proteins, indicating an electrostatic interaction which is disrupted by salt. Hence, the major interaction occurs between the AA- and K-boxes of eIF5-CTD and eIF2-β(1–192) which are both highly charged at neutral pH. The dissociation under salt concentrations below the physiological level is surprising and could be evoked by the fact that only domains and not the full length proteins were used for these studies. Within the MFC, the interaction of the two proteins is further strengthened by the interplay of other eIFs with the eIF5eIF2-β complex. Similar observations were made by others11 for similar eIF5 and eIF2-β constructs. It should be noted that dissociation constants in the μM range have been reported,17 however, these data appear to be of low accuracy. Structure determination and three-dimensional structure The eIF5-CTD crystals were rather small and radiation sensitive. Since selenomethionine-labeled protein yielded crystals of inferior quality, heavyatom derivatives were produced. Soaking of crys-

tals with mercury(II)-acetate and gold(I)-cyanide gave non-isomorphous derivatives with two lowoccupancy binding sites, The resulting electron density maps were not interpretable. Potassium dicyano-aureate(I) resulted in a derivative with one strongly occupied site. Due to the radiation sensitivity a two-wavelength MAD experiment was performed at the LI-remote and LIII-inflection point, in order to maximize the dispersive and Friedel pair differences. A high quality electron density map was obtained by this procedure. As listed in Table 1 two independent MAD experiments on different crystals (xtal 1 and xtal 2) were performed during the process of structure solution. Both data sets yielded the same results and could be used for structure solution. The resulting initial model was later transformed into the native highresolution crystal form and refined at 1.8 ) resolution with good statistics (Table 1). The molecule, which comprises residues 225–407 plus a C-terminal hexa-histidine tag, is generally well defined in the electron density map with exception of the his-tag, amino acids 225–232 and a part of AA-box2, residues 385–399. This latter loop was modeled using the loop-building routine in MODELLER.18,19 The domain has a globular allhelical fold comprising 11 α-helices with approximate dimensions of 35 × 35 × 30 ) (Figure 3). This fold resembles various HEAT repeat proteins. A

Crystal Structure of eIF5-CTD

460 Table 1. Data collection and refinement statistics Native Data collection Space group Cell dimensions a, b, c ()) Beam line Wavelength Resolution ()) Rsym (%) I/σ(I) Completeness (%) No observations No unique reflect, Refinement Resolution ()) Reflections work set Reflection test set Rwork/Rfree (%) No. atoms Water RMS deviations Bond lengths ()) Bond angles (°)

P212121 32.14, 70.25, 81.71 ID29 (ESRF) 1.007 40–1.80 (1.91–1.80) 6.1 (8.5) 27.6 (18.2) 98.4 (97.4) 104,040 17,547

NaAu(CN)2-xtal 1 P212121

NaAu(CN)2-xtal 2 P212121

32.20, 71.15, 80.52 X06SA (SLS) Inflection Remote 1.0397 0.8550 40–2.97 40–2.46 (3.10–2.97) (2.60–2.46) 4.9 (8.6) 3.9 (7.2) 21.4 (11.9) 23.5 (14.8) 96.4 (72.1) 98.7 (93.2) 26,602 48,023 7131 12,719

32.00, 70.79, 80.65 BM14 (ESRF) Inflection Remote 1.0397 0.8550 40–2.5 40–2.50 (2.60–2.50) (2.60–2.50) 3.8 (6.5) 3.8 (7.8) 27.6 (17.6) 27.3 (15.6) 99.5 (97.9) 99.7 (98.3) 46,707 47,414 12,217 12,242

30–1.80 17,303 1287 18.9/22.7 1505 139 0.006 1.19

DALI search revealed significant structural homology to eIF2B-ε (PDB code 1PAQ,16 Z-score 12.0, the RMS deviation of 136 Cα atoms is 3.1 ), the sequence identity is 16%), the nuclear cap-binding protein Cbp80 (PDB code 1H6K,20 Z-score 10.4, RMS deviation of 135 Cα atoms is 3.4 ), sequence identity 7%) and the middle HEAT domain of eIF4GII (PDB code 1HU3,21 Z-score 5.7, RMS deviation of 103 Cα atoms 3.7 ), sequence identity 6%). HEAT domains are modules mediating protein-protein interactions and this is the common biological background of those proteins. A distinct feature of the human eIF5-CTD is the C-terminal helix α11 which is formed by amino acids 399–407 and is tucked into a groove formed by helices α1, α5 and α6 and the loop connecting α9 and α10. This helix guides the AA-2 box roughly perpendicular across AAbox-1 (Figures 4, 5). It does not occur in yeast and plants which means that there the AA-2 box extends freely from the body of the molecule and is not fixed by its C-terminal end. The electrostatic surface potential (Figure 4B) shows a highly dipolar molecule: a strong negative patch is formed by the two AA-boxes while a positive patch is formed by lysines 361, 365 and 369. These patches are involved in the binding to other eIFs (see below). Binding sites for other eIFs Binding of eIF5 to eIF2 is established by the electrostatic interactions of the lysine-rich K-boxes of eIF2-β and the two AA-boxes of eIF5.17,22 The AA-boxes are located close to each other (Figure 4A), with the stretches of acidic residues running almost perpendicular to each other. C-terminal truncation of yeast eIF5 from Trp381 (human

numbering) eliminates eIF2-binding without affecting eIF5 binding to eIF3.10 Single and multiple mutations of glutamic acid residues in the AA-Boxes of eIF5 clearly effect its interaction to eIF2-β. Moreover, the quadruple mutation of the acidic residues Asp344, Glu347, Glu348 and Glu349 to serines decreases eIF2-binding fivefold but affects eIF3 binding only slightly.6 These four carboxylate groups are located on the ‘‘upper’’ surface as shown in Figure 4B (left), quite central in the negative electrostatic patch. Interestingly, AA-box 2 of eIF5 is also a target for CK2. CK2 phosphorylates Ser-389 and Ser-390 in response to cell cycle progression.23 This phosphorylation is important for the smooth transition of the cells from S to M phase and there is some evidence emerging now that phosphorylation is important for the proper eIF2–eIF5 complex formation. Binding to eIF3c has been mapped by a multiple mutation which alters the positively charged amino acids lysine 357, 360, 361, 365, 369 and Arg382 (Figure 4) to glutamine and thereby abolishing binding of eIF3 and eIF1 to eiF5. Nevertheless, the eIF5–eIF2 interaction seems not to be influenced by this surface modification. All of these amino acids are located beneath AA-box 2 in the ‘‘upper right’’ corner of Figure 4 and form the positive electrostatic potential patch mentioned above. Likewise, mutation of the adjacent residues 326 and 327 (His and Lys in yeast, Gln and Ala in human) reduces eIF3cand eIF1-binding by a factor of two without affecting eIF2 binding. Temperature-sensitive mutations Mutants exhibiting a temperature-sensitive phenotype 17,24 can be mapped onto the three-

Crystal Structure of eIF5-CTD

461

Figure 3. Top: Cartoon representation of eIF5-CTD. Shown is a cartoon in rainbow coloring (N-terminus blue, Cterminus red). AA-box 1 (AA1) and AA-box 2 (AA2) are shown in grey and magenta, respectively. Bottom: Stereo cartoon overlay eIF5-CTD and eIF2B-ε. Shown is an overlay of eIF5-CTD (rainbow-colored, blue (N-terminus) to red (C-terminus) and eIF2B-ε (grey, PDB code 1PAQ). The figure was generated with PYMOL [http://www.pymol.org].

dimensional structure, as shown in Figure 5. Leu270, Leu284, Phe285, Ile293, Leu314, Leu337 Met340 and Trp354 were identified as course for temperature-sensitive mutations. All of these mutations affect the hydrophobic core and hence the stability of the molecule, rather than disturbing binding surfaces.

Materials and Methods Plasmid construction The human eiF5-CTD (amino acids 225–407) was subcloned from a cDNA library by PCR methods using the primers 5′-CAGTGACCATCATATGGTTCTGACACTQ

Crystal Structure of eIF5-CTD

462

Figure 4 (legend on opposite page)

Crystal Structure of eIF5-CTD

463

Figure 5. Mapping of temperature-sensitive mutations. Shown is a stereo trace as in Figure 4(a) (top) with the relevant residues (Leu270, Leu284, Phe285, Ile293, Leu314, Leu337, Met340, Trp354) shown as sticks.

CAGTG-3′ and 5′-TTCCTTTATCGCGGCCGCCTTCGAATACACCACCTC-3′. The construct was cloned into the expression vector pET22b(+) (Novagen) using the NdeI and the NotI sites. This construct possesses a hexa-histidine tag at the C-terminus. Human eIF2-β (amino acids 1–192) was cloned into a pET28a E. coli expression vector (Novagen) by PCR methods using the primers 5′-GGGAATTCCATATGTCTGGGGACG-3′ and 5′-CGCGGATCCTTAATTCTTTTCCCTCAT-3′ (using the NdeI and BamHI sites). This construct possesses an N-terminal histidine tag which can be cleaved of with thrombine. Protein expression and purification Escherichia coli strain C43 was used for protein overexpression. Bacteria were cultivated at 37 °C in LB broth and induced for protein expression with 0.5 mM isopropyl-beta-D-thiogalactopyranoside at an OD600 of app. 0.7. Cell growth was continued over night at 20 °C. Cells were harvested by centrifugation (4500g) at 4 °C and resus-

pended in lysis-buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8.0). Cells were disrupted by a French press (twice) and subsequent sonication for 2 minutes on ice. The debris was removed by centrifugation at 4 °C (50,000g) for 1 h. The supernatant was applied to a 10 ml metal-chelate affinity column (Ni-NTA, Qiagen) and the column was washed with lysis-buffer containing 20 mM imidazole until the baseline at 280 nm had stabilized. eIF5-CTD was eluted with lysis-buffer containing 250 mM imidazole. An ion-exchange column was performed as a second purification step. The sample was loaded onto a 6 ml Resource Q™ column (Amersham Biosciences), equilibrated with buffer A (20 mM Tris-HCl, pH 8.0). The bound protein was eluted with a linear NaCl gradient (0–0.5 M) in the same buffer. The protein was further purified by gel filtration using a Superdex75 (Amersham Bioscience) column equilibrated in buffer G (20 mM Tris-HCl pH 8.0, 100 mM NaCl, 2 mM EDTA, 2 mM DTT, 0.02% NaAc). All purification steps were monitored by SDS-PAGE. The pure protein was concentrated to a final concentration of 10–15 mg/ml. Mass spectrometric analysis and N-

Figure 4. Mapping of eIF binding sites. Top: Stereo trace. The trace is rainbow-colored (N-terminus blue, C-terminus red) with exception of AA-box 1 which is colored in grey and AA-box 2 colored in hotpink. The residues involved in eIF2 binding are shown as sticks in pink color: Asp344, Glu347, Glu348 and Glu349, AA-box 2 residues Trp411 to Glu425 are shown as lines in the same color. The amino acids (Lys357, Lys360, Lys361, Lys365, Lys369 and Arg382) identified to be important for eIF3 binding are shown in purple. Bottom: Electrostatic potential calculated by APBS mapped onto the accessible surface at −10 (red) and +10 (blue) kT/e. Left orientation as in top of the picture, right turned by 90 degrees around the vertical. The highly negative charged eIF2 (left) and the positive charged eIF3 binding regions (right) are indicated by a dashed line.

Crystal Structure of eIF5-CTD

464 terminal sequencing were carried out by the Analytical Research Services of the Chemistry Department, University of Berne. Crystallization Crystals of eIF5-CTD were obtained within 1 day by the hanging-drop vapour-diffusion method. Drops were set up by mixing 2 μl of protein solution (10–15 mg/ml) with 2 μl of reservoir solution (200 mM magnesium chloride, 25% PEG 3350, 100 mM Tris-HCl, pH 8.5) and equilibrated against 0.75 ml reservoir solution at 293 K. Typical crystals were needle-shaped with an average size of 500 × 100 × 50 μm3. They belong to the orthorhombic spacegroup P212121 with cell parameters of a = 32.14 ), b = 70.25 ), c = 81.7 ) and contain one monomer per asymmetric unit. Data collection Crystals were mounted by sequential transfer (5% steps) into the crystallization solution containing 20% glycerol (v/v) and were flash-cooled in a nitrogen stream at 110 K prior data collection. Native Data were collected at beamline ID29 (ESRF, Grenoble, France) (Table 1). MAD Data were collected at the Swiss Light Source at 100 K on beamline X06SAwith a mar225 CCD detector (MAR X-rayresearch, Hamburg, Germany) (Table 1). A second MAD experiments was performed at the Grenoble ESRF, beamline BM14 at 100 K, employing a mar225 CCD detector (MAR X-ray-research, Hamburg, Germany) All datasets were integrated and scaled with XDS.25 Data collection statistics is given in Table 1. Structure solution and refinement Selenomethionine-derivatized protein was produced by the pathway-inhibition method.26 However, the resulting crystals were too small and intergrown. Hence, heavyatom derivative crystals were prepared by soaking the native crystals in mother liquor containing 20 mM potassium dicyanoaurate (I) for 2 days. One gold position was determined by SHELXD.27 Phases were computed using SOLVE28 and improved by RESOLVE.29 Automatic model building was performed by RESOLVE.30 164 out of 194 residues were built by the program. This initial model was transported into the unit cell of the native high-resolution data using MOLREP.31 Further density modification, phase extension and automatic model building was carried out by ArpWarp32 using the resulting model from MOLREP and the native highresolution data set. Refinement was effected using REFMAC.33 Refinement statistics are given in Table 1. Binding assay Interaction of eIF5-CTD and eIF2-β(1–192) was tested by analytical gelfiltration using a Superdex75 (Amersham Bioscience) column equilibrated in buffer B (20 mM TrisHCl pH 8.0, 2 mM EDTA, 2 mM DTT, 0.02% sodium azide, and 20 or 100 mM NaCl). Protein samples of eIF5-CTD and eIF2-β(1–192) were mixed at a concentration of app. 1– 5 mg/ml either in buffer B1 (20 mM Tris-HCl pH 8.0, 20 mM NaCl) or buffer B2 (20 mM Tris-HCl pH 8.0, 100 mM NaCl) and incubated for 1 hour at 4 °C before gelfiltration. Size exclusion chromatography steps were performed at 4 °C and RT. Fractions were analyzed by SDS-PAGE.

Atomic coordinates accession codes Atomic coordinates have been deposited with the RCSB Protein Data Bank and are available under accession code 2iu1.

Acknowledgements This work has been supported by the Swiss National Science Foundation and the Berner Hochschulstiftung. We gratefully acknowledge the help of Clemens Schulze-Briese at beamline X06SA, SLS, PSI Villigen, Martin Walsh at beamline BM14, ESRF, Grenoble, and Gordon Leonard at ID29, ESRF, Grenoble.

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Edited by J. Doudna (Received 15 March 2006; received in revised form 3 May 2006; accepted 10 May 2006) Available online 24 May 2006