Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Crystal Structure of Hyperthermophilic Archaeal Initiation Factor 5A: A Homologue of Eukaryotic Initiation Factor 5A (eIF-5A)

Crystal Structure of Hyperthermophilic Archaeal Initiation Factor 5A: A Homologue of Eukaryotic... Abstract Eukaryotic initiation factor 5A (eIF-5A) is ubiquitous in eukaryotes and archaebacteria and is essential for cell proliferation and survival. The crystal structure of the eIF-5A homologue (PhoIF-5A) from a hyperthermophilic archaebacterium Pyrococcus horikoshii OT3 was determined at 2.0 Å resolution by the molecular replacement method. PhoIF-5A is predominantly composed of β-strands comprising two distinct folding domains, an N-domain (residues 1–69) and a C-domain (residues 72–138), connected by a short linker peptide (residues 70–71). The N-domain has an SH3-like barrel, while the C-domain folds in an (oligonucleotide/oligosaccharide binding) OB fold. Comparison of the structure of PhoIF-5A with those of archaeal homologues from Methanococcus jannaschii and Pyrobaculum aerophilum showed that the N-domains could be superimposed with root mean square deviation (rmsd) values of 0.679 and 0.624 Å, while the C-domains gave higher values of 1.824 and 1.329 Å, respectively. Several lines of evidence suggest that eIF-5A functions as a biomodular protein capable of interacting with protein and nucleic acid. The surface representation of electrostatic potential shows that PhoIF-5A has a concave surface with positively charged residues between the N- and C-domains. In addition, a flexible long hairpin loop, L1 (residues 33–41), with a hypusine modification site is positively charged, protruding from the N-domain. In contrast, the opposite side of the concave surface at the C-domain is mostly negatively charged. These findings led to the speculation that the concave surface and loop L1 at the N-domain may be involved in RNA binding, while the opposite side of the concave surface in the C-domain may be involved in protein interaction. Key words: archaebacteria, crystal structure, initiation factor 5A, Pyrococcus horikoshii. Abbreviations: eIF-5A, eukaryotic initiation factor 5A; MjaIF-5A, initiation factor 5A from Methanococcus jannaschii; NCS, non-crystallographic symmetry; OB-fold, oligonucleotide/oligosaccharide binding fold; PaeIF-5A, initiation factor 5A from Pyrobaculum aerophilum; PEG, polyethylene glycol; PhoIF-5A, initiation factor 5A from Pyrococcus horikoshii; rmsd, root mean square deviation. Received October 4, 2002; accepted October 25, 2002 Eukaryotic initiation factor 5A (eIF-5A), ubiquitous in both eukaryotes and archaebacteria, consists of about 140 amino acid residues and was initially named based on the findings that eIF-5A could be isolated from the ribosome-bound fraction and stimulate the synthesis of methionyl-puromycin (1, 2). However, the role of eIF-5A in translation initiation has been questioned because of a lack of correlation between eIF-5A and general protein synthesis (3). Alternatively, it was proposed that eIF-5A may facilitate protein synthesis by promoting nuclear export of specific mRNAs (4). Furthermore, a recent study has suggested that eIF-5A may facilitate the translation of mRNA species required for programmed cell death (5). eIF-5A is the only cellular protein known to contain a hypusine residue (for review, see Refs. 6 and 7). Hypusine is formed by a series of post-translational reactions. The first reaction leading to the formation of hypusine is catalyzed by deoxyhypusine synthase, an enzyme that adds butylamine to the conserved lysine of eIF-5A to form deoxyhypusine. A subsequent reaction in which deoxyhypusine is converted to hypusine is catalyzed by deoxyhypusine hydroxylase (8–10). Hypusine formation is tightly coupled to cell proliferation and is essential for cell survival (11, 12). Disruption of either eIF-5A or the deoxyhypusine synthesis gene in yeast leads to a lethal phenotype (13, 14). Recently, it was reported that hypusine is required for a sequence-specific interaction of eIF-5A with RNA (15). Although eIF-5A is essential for cell proliferation and survival, the precise physiological function of eIF-5A is still unclear. In this study, we determined the crystal structure of an eIF-5A homologue (PhoIF-5A) from the hyperthermophilic archaebacterium Pyrococcus horikoshii OT3 in the hopes that it would be of help in understanding the cellular function and role of eIF-5A. P. horikoshii OT3 isolated from the hot waters of the Okinawa Trench can grow at high temperatures, favoring a temperature of 98°C. The entire length of the genome is 1.74 million base pairs, comprising 2,061 open reading frames. The functions of about 27% of the P. horikoshii gene products can be assigned based on their amino acid sequence analysis (16). A gene (ID code PH1381) product was assigned as the homologue of eIF-5A, sharing 22% identical residues with Saccharomyces cerevisiae eIF-5A. Here, we report the three-dimensional structure of PhoIF-5A determined at 2.0 Å resolution by the molecular replacement method using eIF-5A homologues from Methanococcus jannaschii (PDB code 2EIF) (17) and Pyrobaculum aerophilum (PDB code 1BKB) (18) as search models. MATERIALS AND METHODS Materials Restriction enzymes were purchased from MBI Fermentas. The oligonucleotide primers and thermo sequenase fluorescent labeled primer cycle sequencing kit containing 7-deaza-dGTP were obtained from Amersham Pharmacia Biotech. Plasmid pGEM T-vector and expression plasmid pET-22b were obtained from Promega and Novagen, respectively. Escherichia coli strains JM109 and BL21 (DE3) were used as host cells for cloning and producing recombinant protein, respectively. Polyethylene glycol 4000 (PEG 4000) was obtained from Fluka Chemicals; reagents were purchased at the highest purity available. Cloning, Overproduction, and Purification The gene (ID code PH1381) encoding the P. horikoshii IF-5A (PhoIF-5A) was amplified by PCR and placed under control of the T7 phage promoter on the expression plasmid pET-22b (19). Expression was induced with IPTG, and the resulting protein was purified to apparent homogeneity by ion-exchange chromatography on S-Sepharose. Crystallization and Data Collection The purified protein PhoIF-5A was concentrated to 10 mg ml–1 in 0.1 M Hepes-NaOH buffer (pH 7.5). Crystallization attempts were made by the hanging-drop vapor diffusion method at 18°C using crystal screen kits (Hampton Research) as reservoir solutions. Each drop was formed by mixing equal volumes (2 µl:2 µl) of protein and reservoir solution. Crystals of PhoIF-5A were obtained under conditions of 10% isopropanol and 20% PEG 4000 in one week with a size of 0.1 × 0.1 × 0.2 mm3. For data collection, the crystals of PhoIF-5A were transferred into a cryoprotectant solution containing 20% sucrose in reservoir solution, then mounted in a nylon loop and flash-frozen in a nitrogen stream at 100 K. X-ray diffraction data were collected on beamline BL41XU at SPring-8 of Japan, integrated, scaled and merged by the MOSFLM (20) and SCALA (21) programs. A crystal of PhoIF-5A diffracts to 2.0 Å and belongs to space group P32 with cell dimensions of a = b = 93.3 Å, c =39.4 Å, and γ = 120°. Crystals of PhoIF-5A contain three molecules per asymmetric unit, corresponding to a VM of 2.16 Å3 Da–1. Assuming a value of 0.74 cm3 g–1 for the protein partial specific volume (22), the calculated solvent content in the crystal is 43.1%. The statistics of data collection are summarized in Table 1. Structure Determination and Refinement The crystal structure of PhoIF-5A was determined by the molecular replacement method using the program MOLREP (23). Two search models of archaeal homologues from M. jannaschii (ID: 2EIF) and P. aerophilum (ID: 1BKB), which have 46.4 and 48% identical residues with PhoIF-5A, respectively, were constructed based on the alignment of amino acid sequences. Structure refinement was performed using the program CNS (24), and the 10% reflection data were set aside for the calculation of the free R-factor to monitor the refinement. After the first step of the refinement at 2.5 Å resolution, a model of PhoIF5A was rebuilt using the program O (25). Non-crystallographic symmetry (NCS) restraints were applied throughout the refinement. The coordinates have been deposited in the Protein Data Bank (ID: 1IZ6). RESULTS AND DISCUSSION Structure Determination and Quality In spite of the presence of three independent molecules (MolA, MolB, and MolC) in an asymmetry unit, the self-rotation function did not reveal any local symmetry. However, the native Patterson function that was subsequently calculated showed a peak at translation vector [0.67, 0.33, 0.43] (fractional coordinate), which means that at least two molecules have nearly identical orientations in the asymmetric unit. Table 2 shows the results of the molecular replacement calculated by the program MOLREP. The best solution was obtained from space group P32 and a search model of 1BKB, which gave a correlation coefficient of 40.3% and an R factor of 52.5%. Two of the three independent molecules have nearly identical orientations with a shift vector of [0.67, 0.33, 0.43]. The initial model constructed from 1BKB contained 134 residues (out of a total 136 residues) of which 62 residues possessed full side chains. After generating all side chains by computer, the first round of refinement, including rigid-body, positional, temperature and simulated annealing, was carried out at 2.5 Å resolution, which gave free R- and R-factors of 42.9 and 33.4%, respectively. The structure was remodeled using program O. After several rounds of refinement and manual fitting, the structure was refined to the free R- and R-factors of 23.7 and 19.9%, respectively, at 2.0 Å resolution. The refined structure contains 407 residues [136 (MolA), 136 (MolB), and 135 (MolC)] and 278 water molecules in the asymmetric unit. The N-terminal Met and C-terminal Gln residues are disordered, and the loop L1 (residues 33–41) has weak density. The model has geometries close to ideal with rmsd of 0.009 Å and 1.5° from ideal values for bond lengths and angles, respectively. When the structure was checked using PROCHECK (26), 91.2% of the non-glycine and non-proline residues fell in the most favored regions and 8.8% in the additional allowed region of the Ramachandran plot. The refinement statistics are summarized in Table 3. Overall Structure The refined model of PhoIF-5A is a beta-rich structure with 52.9% β-strand, 8.0% helical (including 310-helix), 18% turn, and 27.1% unclassified coil. PhoIF-5A consists of two distinct domains, an N-domain (residues 1–69) and a C-domain (residues 72–138) (Fig. 1). The secondary structure of PhoIF-5A as defined by the program DSSP (27) is shown in Fig. 2. The N-domain has an SH3-like barrel motif consisting of a 310-helix (α1) and six-stranded (β1, β2, β3, β4, β5, and β6) anti-parallel highly twisted β-sheets. The N-domain has a long hairpin loop, L1 (residues 33–41), including a specific residue, Lys37, that is supposed to be modified to hypusine residue. A quarter of the long β3 strand forms hydrogen bonds with β2, and the remaining part with β4. The β2 strand and the N-terminal portion of β3 are approximately perpendicular to strands β3, β4, β5, and β6. This highly twisted β-sheet forms a distorted β-barrel with a short 310-helix on the top. Further, hydrophobic residues Val7, Val9, Leu12, Ile18, Ile20, Ile27, Val32, Ala43, Ile45, Ile58, Val66, and Val68 form a hydrophobic core inside the β-barrel. The C-domain has an oligonucleotide/oligosaccharide binding fold (OB-fold), comprising two short α-helices (α2 and α3) and a five-stranded (β7, β8, β9, β10, and β11) anti-parallel β-barrel arranged differently from the N-terminal β-barrel. The top of C-terminal β-barrel is capped by a loop between α3 and β10. The long β7 strand forms hydrogen bonds with β8 at the center of the peptide chain. Like the N-domain, the C-terminal β-barrel also forms a hydrophobic core with residues Ala76, Val78, Ile81, Val86, Ile88, Phe97, Val99, Ile101, Val105, Leu113, Val119, and Ile130. The two domains of PhoIF-5A are connected by a peptide linker (residues 70–71), which joins the last β-strand, β6, of N-domain and the first β-strand, β7, of the C-domain. The interface of the two domains is formed by residues from β2, the loop between β4 and β5, and β10. The contact between the two domains involves several hydrogen bonds and a hydrophobic core including residues Tyr17, Pro24, Ile50, Phe51, Ile71, Trp122, Thr124, and Leu125. PhoIF-5A has a concave surface between the N- and C-domains with an approximate size of 6.5 Å × 6 Å × 15 Å calculated from the three independent molecules (MolA, MolB, and MolC) in the asymmetry unit. Structure Comparison The amino acid sequence of PhoIF-5A was aligned with those of two archaeal homologues as well as eukaryotic homologues using the program CLUSTAL W (28), as shown in Fig. 2. Most of the conserved sequences are within the N-domain, while the C-domain is less conserved. The overall structure of PhoIF-5A displays significant similarity to those of archaeal homologues (Fig. 3a). Superposition of the entire PhoIF-5A with the two homologues PaeIF-5A and MjaIF-5A using the program LSQABK (29) gave rmsd values of 1.276 and 1.690 Å, respectively, for 117 Cα atoms. When independent domains were superimposed, the N-domains of the three archaeal IF-5As have a very fixed structure (except loop L1) as shown by the small rmsd values (0.624 and 0.679 Å), whereas the rmsd values of the C-domains are 1.329 and 1.824 Å (Table 4). Distinct rmsd values for the two domains were also obtained by comparison of the three independent molecules of PhoIF-5A in the asymmetric unit, as shown in Fig. 3b and Table 4, suggesting inter-domain movement within the molecule. Thus, the peptide linker of the two domains mediates an inter-domain flexibility of about 7°. Possible RNA-Binding Site The N-domain of PhoIF-5A contains an SH3-like barrel motif, while the C-domain folds in an OB-fold. The DALI-server (30) shows that SH3-like barrel motifs are found in the DNA binding domain of HIV-integrase (31), the repressor of the E. coli biotin biosynthetic operon (32), and the C-terminal domain of the ribosomal protein L2-RBD (33). On the other hand, the OB-fold is one of the most common RNA binding modules and is found in several RNA binding proteins that are known to be associated with translation, including initiation factors, such as IF-1 (34) and eIF-2α (35), ribosomal proteins, such as S1 (36), S17 (37), and L2 (33), and the N-terminal domain of aspartyl tRNA synthetase (38). Interestingly, the ribosomal protein L2-RBD, like eIF-5A, has an SH3-like barrel motif and an OB-fold joined by a flexible peptide (33). However, the N-terminal domain of L2-RBD is an OB-fold and the C-terminal domain is an SH3-like barrel, the reverse of the situation in eIF-5A. Nevertheless, these findings strongly suggest that eIF-5A may interact with nucleic acids, particularly RNA. Indeed, it is known that eIF-5A is capable of binding to the Rev response element and U6 RNA in vitro (39). Furthermore, Xu and Chan reported that yeast eIF-5A specifically binds RNA containing the consensus sequence AAAUGUCACAC (15). It is generally known that the electrostatic interactions between exposed positively charged residues and the phosphate backbone of RNA, and stacking interactions between exposed aromatic side chains and non-base-paired RNA nucleotides are common features of protein-RNA interactions (40). As for the electrostatic potential of PhoIF-5A, the concave surface between the N- and C-domains is positively charged, as depicted in Fig. 4. Positively charged residues Lys13, Arg16, and Arg26 in the N-domain, and Lys73 and Arg132 in the C-domain are clustered at the interface of the two domains (Fig. 5). Among them, Lys13 and Lys73 are highly conserved as positive charged residues (either lysine or arginine) in five eIF-5As including yeast and human eIF-5A. In addition, two highly conserved aromatic residues, Tyr17 and Phe51, are located at the valley between the N- and C-domains. These structural features, including inter-domain flexibility, suggest that the concave surface between the N- and C-domains may present a potential surface for interaction with RNA, and that the inter-domain flexibility may become ordered upon RNA binding. It has been reported that hypusine formation is tightly coupled to cell proliferation and is essential for cell survival (6, 7, 11). Disruption of either the eIF-5A or deoxyhypusine synthase gene in yeast leads to a lethal phenotype (12–14). Further, it has been reported that this modification is required for the RNA binding activity of eIF-5A (15). Apparently, a tip of the loop L1 is positively charged, due to the Lys37 and His38 residues. Modification of Lys37 to hypusin further increases the positive charge. It is thus assumed that the tip of loop L1 may be another RNA binding site in eIF-5A, although the possibility that the modification of Lys37 could cause a structural change that results in the formation of an RNA binding site(s) in eIF-5A cannot be excluded. The unambiguous assignment of the RNA binding site must await the determination of the crystal structure of eIF-5A in complex with RNA containing the consensus sequence (15). In contrast, the molecular surface of the opposite side of the concave surface in the C-domain is highly negatively charged, as indicated in Fig. 4. It is thus unlikely that the opposite side of the concave surface of the C-domain interacts directly with RNA. It has been proposed that eIF-5A functions as a bimodular protein, capable of interacting with both protein and nucleic acid (39). Indeed, it has been reported that eIF-5A interacts with several proteins, including deoxyhypusine synthase (41), transglutaminase II (42), L5 (43), CRM1 (44), and exportin 4 (45). It is thus tempting to speculate that eIF-5A may function as a bimodular protein, with RNA binding sites at the concave surface and loop L1, and protein binding sites at the C-domain. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant from the National Project on Protein Structural and Functional Analyses. We thank Dr. M. Kawamoto of the SPring-8, Japan, for his help in data collection and Dr. Y. Kakuta for valuable comments and suggestions on the manuscript. + To whom correspondence should be addressed. Tel/Fax: +81-92-642-2853, E-mail: mkimura@agr.kyushu-u.ac.jp View largeDownload slide Fig. 1. AstereoscopicdrawingofthetranslationinitiationfactorPhoIF-5AfromP.horikoshii. The balls-and-sticks represent the specific amino acid Lys37 that is supposed to be modified to hypusine. View largeDownload slide Fig. 1. AstereoscopicdrawingofthetranslationinitiationfactorPhoIF-5AfromP.horikoshii. The balls-and-sticks represent the specific amino acid Lys37 that is supposed to be modified to hypusine. View largeDownload slide Fig. 2. Sequencecomparisonofthetranslationinitiationfactor5As. The alignment of amino acid sequences was first calculated by CLUSTAL W (28) and modified based on the secondary structures of PhoIF-5A (present work), MjaIF-5A (17), and PaeIF-5A (18). The amino acid residues are shown as follows: completely identical (pink), conserved change (yellow), completely identical only in archaeal IF-5As (light blue). The secondary structure elements are defined by the DSSP program (27). Lys37, which is modified to hypusine, is marked by an asterisk. View largeDownload slide Fig. 2. Sequencecomparisonofthetranslationinitiationfactor5As. The alignment of amino acid sequences was first calculated by CLUSTAL W (28) and modified based on the secondary structures of PhoIF-5A (present work), MjaIF-5A (17), and PaeIF-5A (18). The amino acid residues are shown as follows: completely identical (pink), conserved change (yellow), completely identical only in archaeal IF-5As (light blue). The secondary structure elements are defined by the DSSP program (27). Lys37, which is modified to hypusine, is marked by an asterisk. View largeDownload slide Fig. 3. StructuralcomparisonofthearchaealIF-5As. a: Superimposition of the backbone atoms of three archaeal homologues, PhoIF-5A (MolA, red), PaeIF-5A (green) (18), and MjaIF-5A (yellow) (17). b: Superimposition of the backbone atoms of three independent copies in the asymmetric unit of PhoIF-5A; MolA (red), MolB (green), and MolC (yellow). View largeDownload slide Fig. 3. StructuralcomparisonofthearchaealIF-5As. a: Superimposition of the backbone atoms of three archaeal homologues, PhoIF-5A (MolA, red), PaeIF-5A (green) (18), and MjaIF-5A (yellow) (17). b: Superimposition of the backbone atoms of three independent copies in the asymmetric unit of PhoIF-5A; MolA (red), MolB (green), and MolC (yellow). View largeDownload slide Fig. 4. SurfacerepresentationoftheelectrostaticpotentialofPhoIF-5A. Surface representation of the electrostatic potential of PhoIF-5A was calculated by GRASP (46). The surface potential is displayed as a color gradient from red (negative) to blue (positive). Each view is rotated by 90°. View largeDownload slide Fig. 4. SurfacerepresentationoftheelectrostaticpotentialofPhoIF-5A. Surface representation of the electrostatic potential of PhoIF-5A was calculated by GRASP (46). The surface potential is displayed as a color gradient from red (negative) to blue (positive). Each view is rotated by 90°. View largeDownload slide Fig. 5. AclusterofpositivelychargedresiduesattheputativeRNA-bindingregionofPhoIF-5A. a: The balls-and-sticks represent positive charged residues that may contribute to RNA binding. b: View rotated 90° from (a). View largeDownload slide Fig. 5. AclusterofpositivelychargedresiduesattheputativeRNA-bindingregionofPhoIF-5A. a: The balls-and-sticks represent positive charged residues that may contribute to RNA binding. b: View rotated 90° from (a). Table 1. Summary of data collection. Space group  P32  Unit cell  a = b = 93.3 Å, c = 39.2 Å, γ = 120°  Z′a  3 (VM = 2.16 Å3/Da)  Wavelength (Å)  0.90  Resolution (Å)b  100–2.0 (2.11–2.0)  Number of observed reflections  148,604  Unique reflections  25,949  Completeness (%)  100 (99.9)  Averaged redundancy  5.7 (5.7)  Averaged I/σ (I)  8.5 (2.4)  Rmeans (%)c  8.1 (34.9)  Space group  P32  Unit cell  a = b = 93.3 Å, c = 39.2 Å, γ = 120°  Z′a  3 (VM = 2.16 Å3/Da)  Wavelength (Å)  0.90  Resolution (Å)b  100–2.0 (2.11–2.0)  Number of observed reflections  148,604  Unique reflections  25,949  Completeness (%)  100 (99.9)  Averaged redundancy  5.7 (5.7)  Averaged I/σ (I)  8.5 (2.4)  Rmeans (%)c  8.1 (34.9)  aZ′ is the number of molecules in an asymmetric unit. bValues in parentheses are for the outermost resolution shell. cRmeans = Σh[m/(m – 1)]1/2Σj|<I>h – Ih,j|ΣhΣjIh,j, where <I>h is the mean intensity of symmetry-equivalent reflections and m is redundancy. View Large Table 2. Cross-rotation and translation function statistics. Model    θ1 (°)  θ2 (°)  θ3(°)  x  y  z  CC (%)  R (%)    Cross rotation function (P3x)  1BKB  answer1  22.79  67.92  –81.54        4.56      answer2  87.68  57.23  74.66        4.36      answer3  44.06  104.45  37.07        4.02      answer4  39.06  37.91  –80.41        3.73      answer5  57.39  69.98  183.05        3.72    2EIF  answer1  58.87  107.00  32.9        4.38      answer2  82.72  100.71  66.89        4.09      answer3  75.60  72.56  119.94        4.08      answer4  65.06  108.67  45.80        3.88      answer5  4.33  46.90  127.29        3.80      Translation function  1BKB  P32                    Third site using two sites of (22.79, 67.92, –81.54, 0.167, 0.118, 0.000) and (32.69, 38.81, –75.54, 0.797, 0.448, 0.334)    answer1  39.06  37.91  –80.41  0.462  0.789  0.771  40.5  52.5    answer2  39.06  37.91  –80.41  0.381  0.777  0.044  36.3  54.4    answer3  39.06  37.91  –80.41  0.477  0.688  0.815  36.1  54.0    P31                    Third site using two sites of (22.79, 67.92, –81.50, 0.607, 0.288, 0.000) and (39.06, 37.91, –80.41, 0.189, 0.239, 0.695)    answer1  22.79  67.92   –81.54  0.277  0.624  0.431  31.4  56.9    answer2  22.79  67.92  –81.54  0.218  0.144  0.036  30.3  56.7    answer3  22.79  67.92  –81.54  0.099  0.788  0.944  30.3  56.8  2EIF  P32                    Third site using two sites of (58.87, 107.00, 32.95, 0.878, 0.050, 0.000) and (82.72, 100.71, 66.89, 0.544, 0.345, 0.332)    answer1  82.72  100.71  66.89  0.203  0.674  0.758  36.9  54.6    answer2  82.72  100.71  66.89  0.207  0.672  0.649  32.7  55.8    answer3  82.72  100.71  66.89  0.194  0.643  0.874  32.6  55.6    P31                    Third site using two sites of (58.87, 107.00, 32.95, 0.377, 0.284, 0.000) and (82.72, 100.71, 66.89, 0.056, 0.362, 0.584)    answer1  32.38  50.18  –65.46  0.394  0.421  0.082  31.5  55.8    answer2  32.38  50.18  –65.46  0.713  0.471  0.874  30.7  56.2    answer3  32.38  50.18  –65.46  0.200  0.295  0.534  30.7  56.3  Model    θ1 (°)  θ2 (°)  θ3(°)  x  y  z  CC (%)  R (%)    Cross rotation function (P3x)  1BKB  answer1  22.79  67.92  –81.54        4.56      answer2  87.68  57.23  74.66        4.36      answer3  44.06  104.45  37.07        4.02      answer4  39.06  37.91  –80.41        3.73      answer5  57.39  69.98  183.05        3.72    2EIF  answer1  58.87  107.00  32.9        4.38      answer2  82.72  100.71  66.89        4.09      answer3  75.60  72.56  119.94        4.08      answer4  65.06  108.67  45.80        3.88      answer5  4.33  46.90  127.29        3.80      Translation function  1BKB  P32                    Third site using two sites of (22.79, 67.92, –81.54, 0.167, 0.118, 0.000) and (32.69, 38.81, –75.54, 0.797, 0.448, 0.334)    answer1  39.06  37.91  –80.41  0.462  0.789  0.771  40.5  52.5    answer2  39.06  37.91  –80.41  0.381  0.777  0.044  36.3  54.4    answer3  39.06  37.91  –80.41  0.477  0.688  0.815  36.1  54.0    P31                    Third site using two sites of (22.79, 67.92, –81.50, 0.607, 0.288, 0.000) and (39.06, 37.91, –80.41, 0.189, 0.239, 0.695)    answer1  22.79  67.92   –81.54  0.277  0.624  0.431  31.4  56.9    answer2  22.79  67.92  –81.54  0.218  0.144  0.036  30.3  56.7    answer3  22.79  67.92  –81.54  0.099  0.788  0.944  30.3  56.8  2EIF  P32                    Third site using two sites of (58.87, 107.00, 32.95, 0.878, 0.050, 0.000) and (82.72, 100.71, 66.89, 0.544, 0.345, 0.332)    answer1  82.72  100.71  66.89  0.203  0.674  0.758  36.9  54.6    answer2  82.72  100.71  66.89  0.207  0.672  0.649  32.7  55.8    answer3  82.72  100.71  66.89  0.194  0.643  0.874  32.6  55.6    P31                    Third site using two sites of (58.87, 107.00, 32.95, 0.377, 0.284, 0.000) and (82.72, 100.71, 66.89, 0.056, 0.362, 0.584)    answer1  32.38  50.18  –65.46  0.394  0.421  0.082  31.5  55.8    answer2  32.38  50.18  –65.46  0.713  0.471  0.874  30.7  56.2    answer3  32.38  50.18  –65.46  0.200  0.295  0.534  30.7  56.3  View Large Table 3. Final refinement statistics. Resolution range (Å)  10.0–2.0  Number of reflections  25,746  Completeness (%)  100  Total number of non-hydrogen atoms     Protein  3,158   Solvent  279  R-factor (%)a  18.5  R-free-factor (%)b  23.6  rmsd deviation from standard values     Bond lengths (Å)  0.009   Bond angles (deg)  1.628  Average B-factor (Å2)     Protein  27.7   Solvent  37.4  Ramachandran plotc     Residues in most favored regions (%)  91.2   Residues in additional allowed regions (%)  8.8  Resolution range (Å)  10.0–2.0  Number of reflections  25,746  Completeness (%)  100  Total number of non-hydrogen atoms     Protein  3,158   Solvent  279  R-factor (%)a  18.5  R-free-factor (%)b  23.6  rmsd deviation from standard values     Bond lengths (Å)  0.009   Bond angles (deg)  1.628  Average B-factor (Å2)     Protein  27.7   Solvent  37.4  Ramachandran plotc     Residues in most favored regions (%)  91.2   Residues in additional allowed regions (%)  8.8  aR-factor = Σ|Fobs – Fcal|/ΣFobs, where Fobs and Fcal are observed and calculated structure factor amplitudes. bR-free-factor value was calculated for R-factor, using only an unrefined subset of reflections data (10%). cRamachandran plot was calculated by PROCHECK (26). View Large Table 4. Superposition of IF-5As against PhoIF-5A (MolA).   rmsda    Fitting by N-domainb    Fitting by C-domainc  Fitting by alld    N-domain  C-domain    N-domain  C-domain  PhoIF-5A (MolB)  0.447  2.754    3.861  0.989  1.216  PhoIF-5A (MolC)  0.566  2.424    2.977  0.968  1.088  PaeIF-5A  0.624  2.325    3.740  1.329  1.276  MjaIF-5A  0.679  3.845    4.049  1.824  1.690    rmsda    Fitting by N-domainb    Fitting by C-domainc  Fitting by alld    N-domain  C-domain    N-domain  C-domain  PhoIF-5A (MolB)  0.447  2.754    3.861  0.989  1.216  PhoIF-5A (MolC)  0.566  2.424    2.977  0.968  1.088  PaeIF-5A  0.624  2.325    3.740  1.329  1.276  MjaIF-5A  0.679  3.845    4.049  1.824  1.690  armsd = (Σi(xi,molecule1 – xi,molecule2)2/N)1/2, xi = (xi, yi, zi). brmsd values are calculated using Cα atoms of 59 residues (without loop1) and 58 residues in N- and C-domain, respectively, after fitting by N-domain. crmsd values are calculated using Cα atoms of 59 residues (without loop1) and 58 residues in N- and C-domain, respectively, after fitting by C-domain. drmsd values are calculated using Cα atoms of 117 residues. View Large References 1. Kemper, W.M., Berry, K.W., and Merrick, W.C. ( 1976) Purification and properties of rabbit reticulocyte protein synthesis initiation factors M2Balpha and M2Bbeta. J. Biol. Chem.  251, 5551–5557 Google Scholar 2. Benne, R. and Hershey, J.W. ( 1978) The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes. J. Biol. Chem.  253, 3078–3087 Google Scholar 3. Kang, H.A. and Hershey, J.W.B. ( 1994) Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae. J. Biol. Chem.  269, 3934–3940 Google Scholar 4. Bevec, D. and Hauber, J. ( 1997) Eukaryotic initiation factor 5A activity and HIV-Rev function. Biol. Signals  6, 124–133 Google Scholar 5. Wang, T.-W., Ku, L., Wang, D., and Thompson, J.E. ( 2001) Isolation and characterization of senescence-induced cDNA encoding deoxyhypusine synthase and eukaryotic translation initiation factor 5A from tomato. J. Biol. Chem.  276, 17541–17549 Google Scholar 6. Park, M.H., Wolff, E.C., and Folk, J.E. ( 1993) Hypusine: its post-translational formation in eukaryotic initiation factor 5A and its potential role in cellular regulation. Biofactors  4, 95–104 Google Scholar 7. Chen, K.Y. and Liu, A.Y. ( 1997) Biochemistry and function of hypusine formation on eukaryotic initiation factor 5A. Biol. Signals  6, 105–109 Google Scholar 8. Park, M.H., Cooper, H.I., and Folk, J.E. ( 1982) The biosynthesis of protein-bound hypusine (N-epsilon-(4-amino-2-hydroxybutyl) lysine). Lysine as the amino acid precursor and the intermediate role of deoxyhypusine (N-epsilon-(4-aminobutyl) lysine). J. Biol. Chem.  257, 7219–7222 Google Scholar 9. Park, M.H. and Wolff, E.C. ( 1988) Cell-free synthesis of deoxyhypusine. Separation of protein substrate and enzyme and identification of 1,3-diaminopropane as a product of spermidine cleavage. J. Biol. Chem.  263, 15264–15269 Google Scholar 10. Wolff, E.C., Park, M.H., and Folk, J.E. ( 1990) Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD. J. Biol. Chem.  265, 4793–4799 Google Scholar 11. Park, M.H., Lee, Y.B., and Joe, Y.A. ( 1997) Hypusine is essential for eukaryotic cell proliferation. Biol. Signals  6, 115–123 Google Scholar 12. Park, M.H., Joe, Y.A., and Kang, K.R. ( 1998) Deoxyhypusine synthase activity is essential for cell viability in the yeast Saccharomyces cerevisiae. J. Biol. Chem.  273, 1677–1683 Google Scholar 13. Schnier, J., Schwelberger, H.G., Smit-McBride, Z., Kang, H.A., and Hershey, J.W.B. ( 1991) Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol.  11, 3105–3144 Google Scholar 14. Sasaki, K., Abid, M.R., and Miyazaki, M. ( 1996) Deoxyhypusine synthase gene is essential for cell viability in the yeast Saccharomyces cerevisiae. FEBS Lett.  384, 151–154 Google Scholar 15. Xu, A. and Chen, K.Y. ( 2001) Hypusine is required for a sequence-specific interaction of eukaryotic initiation factor 5A with postsystematic evolution of ligands by exponential enrichment RNA. J. Biol. Chem.  276, 2555–2561 Google Scholar 16. Kawarabayashi, Y., Sawada, M., Horikawa, H., Haikawa, Y., Hino, Y., Yamamoto, S., Sekine, M., Baba, S., Kosugi, H., Hosoyama, A., Nagai, Y., Sakai, M., Ogura, K., Otsuka, R., Nakazawa, H., Takamiya, M., Ohfuku, Y., Funahashi, T., Tanaka, T., Kudoh, Y., Yamazaki, J., Kushida, N., Oguchi, A., Aoki, K., and Kikuchi, H. ( 1998) Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. DNA Res.  5, 55–76 Google Scholar 17. Kim, K.K., Hung, L-W., Yokota, H., Kim, R., and Kim, S-H. ( 1998) Crystal structure of eukaryotic translation initiation factor 5A from Methanococcus jannaschii at 1.8 Å resolution. Proc. Natl. Acad. Sci. USA  95, 10419–10424 Google Scholar 18. Peat, T.S., Newman, J., Waldo, G.S., Berendzen, J., and Terwilliger, T.C. ( 1998) Structure of translation initiation factor 5A from Pyrobaculum aerophilum at 1.75 Å resolution. Structure  6, 1207–1214 Google Scholar 19. Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. ( 1990) Use of T7 RNA polymerase to direct the expression of cloned genes. Methods Enzymol.  185, 60–89 Google Scholar 20. Leslie, A.G.W. ( 1993) Auto-indexing of rotation diffraction images and parameter refinement in Proceeding of the CCP4 Study Weekend (Sawyer, L., Isaacs, N., and Bailey, S., eds.) pp. 44–51, Daresbury Laboratory, Daresbury, UK Google Scholar 21. Evans, P.R. ( 1997) Scaling of MAD data in Proceeding of the CCP4 Study Weekend (Wilson, K.S., Davies, G., Ashton, A.W., and Bailey, S., eds.) pp. 97–102, Daresbury Laboratory, Daresbury, UK Google Scholar 22. Mattews, B.W. ( 1968) Solvent content of protein crystal. J. Mol. Biol.  33, 491–497 Google Scholar 23. Vagin, A. and Teplyakov, A. ( 1997) MOLREP: An automated program for molecular replacement. J. Appl. Crystallogr.  30, 1022–1025 Google Scholar 24. Brunger, A.T., Adams, P.D., Clore, G.M., Delano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jlang, J-S., Kuszewski, J., Nilges, N., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T., and Warren, G.L. ( 1998) Crystallography and NMR System (CNS): A new software system for macromolecular structure determination. Acta Crystallogr. D   54, 905–921 Google Scholar 25. Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. ( 1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A   47, 110–119 Google Scholar 26. Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. ( 1993) PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr.  26, 283–291 Google Scholar 27. Kabsch, W. and Sander, C. ( 1983) Dictionary of protein secondary structures: Pattern recognition of hydrogen-bonded and geometrical features. Biopolymers  22, 2577–2637 Google Scholar 28. Thompson, J.D., Higgins, D.G., and Gibson, T.J. ( 1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res.  22, 4673–4680 Google Scholar 29. Kabsch, W. ( 1976) A solution for the best rotation to relate two sets of vectors. Acta Cryst. A  32, 922–923 Google Scholar 30. Holm, L. and Sander, C. ( 1993) Protein structure comparison by alignment of distance matrices. J. Mol. Biol.  233, 123–138 Google Scholar 31. Lodi, P.J., Ernst, J.A., Kuszewski, J., Hickman, A.B., Engelman, A., Craigie, R., Clore, G.M., and Gronenborn, A.M. ( 1995) Solution structure of the DNA binding domain of HIV-1 integrase. Biochemistry  34, 9826–9833 Google Scholar 32. Wilson, K.P., Shewchuk, L.M., Brennan, R.G., Otsuka, A.J., and Mathews, B.W. ( 1992) Escherichia coli biotin hole enzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains. Proc. Natl. Acad. Sci. USA  89, 9257–9261 Google Scholar 33. Nakagawa, A., Nakashima, T., Taniguchi, M., Hosaka, H., Kimura, M., and Tanaka, I. ( 1999) The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome. EMBO J.  18, 1459–1467 Google Scholar 34. Sette, M., van Tilborg, P., Spurio, R., Kaptein, R., Paci, M., Gualerzi, C.O., and Boelens, R. ( 1997) The structure of the translational initiation factor IF1 from E. coli contains an oligomer-binding motif. EMBO J.  16, 1436–1443 Google Scholar 35. Nonato, M.C., Widom, J., and Clardy, J. ( 2002) Crystal structure of the N-terminal segment of human eukaryotic translation initiation factor 2α. J. Biol. Chem.  277, 17057–17061 Google Scholar 36. Bycroft, M., Hubbard, T.J., Proctor, M., Freund, S.M., and Murzin, A.G. ( 1997) The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold. Cell  88, 235–242 Google Scholar 37. Golden, B.L., Hoffman, D.W., Ramakrishnan, V., and White, S.W. ( 1993) Ribosomal protein S17: characterization of the three-dimensional structure by 1H and 15N NMR. Biochemistry  32, 12812–12820 Google Scholar 38. Ruff, M., Krishnaswamy, S., Boeglin, M., Poterszman, A., Mitschler, A., Podjarny, A., Rees, B., Thierry, J.C., and Moras, D. ( 1991) Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp). Science  252, 1682–1689 Google Scholar 39. Liu, Y.P., Nemeroff, M., Yan, Y.P., and Chen, K.Y. ( 1997) Interaction of eukaryotic initiation factor 5A with the human immunodeficiency virus type 1 Rev response element RNA and U6 snRNA requires deoxyhypusine or hypusine modification. Biol. Signals  6, 166–174 Google Scholar 40. Burd, C.G. and Dreyfuss, G. ( 1994) Conserved structures and diversity of functions of RNA-binding proteins. Science  265, 615–621 Google Scholar 41. Lee, Y.B., Joe, Y.A., Wolff, E.C., Dimitriadis, E.K., and Park, M.H. ( 1999) Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor. Biochem. J.  340, 273–281 Google Scholar 42. Singh, U.S., Li, Q., and Cerione, R. ( 1998) Identification of the eukaryotic initiation factor 5A as a retinoic acid-stimulated cellular binding partner for tissue transglutaminase II. J. Biol. Chem.  273, 1946–1950 Google Scholar 43. Schatz, O., Oft, M., Dascher, C., Schebesta, M., Rosorius, O., Jaksche, H., Dobrovnik, M., Bevec, D., and Hauber, J. ( 1998) Interaction of the HIV-1 Rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5. Proc. Natl. Acad. Sci. USA  95, 1607–1612 Google Scholar 44. Elfgang, C., Rosorius, O., Hofer, L., Jaksche, H., Hauber, J., and Bevec, D. ( 1999) Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals. Proc. Natl. Acad. Sci. USA  96, 6229–6234 Google Scholar 45. Lipowsky, G., Bischoff, F.R., Schwarzmaier, P., Kraft, R., Kostka, S., Hartmann, E., Kutay, U., and Gorlich, D. ( 2000) Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes. EMBO J.  19, 4362–4371 Google Scholar 46. Nicholls, A., Sharp, K., and Honig, B. ( 1991) Protein folding and association; insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet.  11, 281–296 Google Scholar Author notes 1Division of Biological Science, Graduate School of Science, Hokkaido University, Sapporo 066-0810; and 2Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Crystal Structure of Hyperthermophilic Archaeal Initiation Factor 5A: A Homologue of Eukaryotic Initiation Factor 5A (eIF-5A)

Download PDF
7 pages

Loading next page...
 
/lp/oxford-university-press/crystal-structure-of-hyperthermophilic-archaeal-initiation-factor-5a-a-NVBBUEW9j7

References (30)

Publisher
Oxford University Press
ISSN
0021-924X
eISSN
1756-2651
DOI
10.1093/jb/mvg011
Publisher site
See Article on Publisher Site

Abstract

Abstract Eukaryotic initiation factor 5A (eIF-5A) is ubiquitous in eukaryotes and archaebacteria and is essential for cell proliferation and survival. The crystal structure of the eIF-5A homologue (PhoIF-5A) from a hyperthermophilic archaebacterium Pyrococcus horikoshii OT3 was determined at 2.0 Å resolution by the molecular replacement method. PhoIF-5A is predominantly composed of β-strands comprising two distinct folding domains, an N-domain (residues 1–69) and a C-domain (residues 72–138), connected by a short linker peptide (residues 70–71). The N-domain has an SH3-like barrel, while the C-domain folds in an (oligonucleotide/oligosaccharide binding) OB fold. Comparison of the structure of PhoIF-5A with those of archaeal homologues from Methanococcus jannaschii and Pyrobaculum aerophilum showed that the N-domains could be superimposed with root mean square deviation (rmsd) values of 0.679 and 0.624 Å, while the C-domains gave higher values of 1.824 and 1.329 Å, respectively. Several lines of evidence suggest that eIF-5A functions as a biomodular protein capable of interacting with protein and nucleic acid. The surface representation of electrostatic potential shows that PhoIF-5A has a concave surface with positively charged residues between the N- and C-domains. In addition, a flexible long hairpin loop, L1 (residues 33–41), with a hypusine modification site is positively charged, protruding from the N-domain. In contrast, the opposite side of the concave surface at the C-domain is mostly negatively charged. These findings led to the speculation that the concave surface and loop L1 at the N-domain may be involved in RNA binding, while the opposite side of the concave surface in the C-domain may be involved in protein interaction. Key words: archaebacteria, crystal structure, initiation factor 5A, Pyrococcus horikoshii. Abbreviations: eIF-5A, eukaryotic initiation factor 5A; MjaIF-5A, initiation factor 5A from Methanococcus jannaschii; NCS, non-crystallographic symmetry; OB-fold, oligonucleotide/oligosaccharide binding fold; PaeIF-5A, initiation factor 5A from Pyrobaculum aerophilum; PEG, polyethylene glycol; PhoIF-5A, initiation factor 5A from Pyrococcus horikoshii; rmsd, root mean square deviation. Received October 4, 2002; accepted October 25, 2002 Eukaryotic initiation factor 5A (eIF-5A), ubiquitous in both eukaryotes and archaebacteria, consists of about 140 amino acid residues and was initially named based on the findings that eIF-5A could be isolated from the ribosome-bound fraction and stimulate the synthesis of methionyl-puromycin (1, 2). However, the role of eIF-5A in translation initiation has been questioned because of a lack of correlation between eIF-5A and general protein synthesis (3). Alternatively, it was proposed that eIF-5A may facilitate protein synthesis by promoting nuclear export of specific mRNAs (4). Furthermore, a recent study has suggested that eIF-5A may facilitate the translation of mRNA species required for programmed cell death (5). eIF-5A is the only cellular protein known to contain a hypusine residue (for review, see Refs. 6 and 7). Hypusine is formed by a series of post-translational reactions. The first reaction leading to the formation of hypusine is catalyzed by deoxyhypusine synthase, an enzyme that adds butylamine to the conserved lysine of eIF-5A to form deoxyhypusine. A subsequent reaction in which deoxyhypusine is converted to hypusine is catalyzed by deoxyhypusine hydroxylase (8–10). Hypusine formation is tightly coupled to cell proliferation and is essential for cell survival (11, 12). Disruption of either eIF-5A or the deoxyhypusine synthesis gene in yeast leads to a lethal phenotype (13, 14). Recently, it was reported that hypusine is required for a sequence-specific interaction of eIF-5A with RNA (15). Although eIF-5A is essential for cell proliferation and survival, the precise physiological function of eIF-5A is still unclear. In this study, we determined the crystal structure of an eIF-5A homologue (PhoIF-5A) from the hyperthermophilic archaebacterium Pyrococcus horikoshii OT3 in the hopes that it would be of help in understanding the cellular function and role of eIF-5A. P. horikoshii OT3 isolated from the hot waters of the Okinawa Trench can grow at high temperatures, favoring a temperature of 98°C. The entire length of the genome is 1.74 million base pairs, comprising 2,061 open reading frames. The functions of about 27% of the P. horikoshii gene products can be assigned based on their amino acid sequence analysis (16). A gene (ID code PH1381) product was assigned as the homologue of eIF-5A, sharing 22% identical residues with Saccharomyces cerevisiae eIF-5A. Here, we report the three-dimensional structure of PhoIF-5A determined at 2.0 Å resolution by the molecular replacement method using eIF-5A homologues from Methanococcus jannaschii (PDB code 2EIF) (17) and Pyrobaculum aerophilum (PDB code 1BKB) (18) as search models. MATERIALS AND METHODS Materials Restriction enzymes were purchased from MBI Fermentas. The oligonucleotide primers and thermo sequenase fluorescent labeled primer cycle sequencing kit containing 7-deaza-dGTP were obtained from Amersham Pharmacia Biotech. Plasmid pGEM T-vector and expression plasmid pET-22b were obtained from Promega and Novagen, respectively. Escherichia coli strains JM109 and BL21 (DE3) were used as host cells for cloning and producing recombinant protein, respectively. Polyethylene glycol 4000 (PEG 4000) was obtained from Fluka Chemicals; reagents were purchased at the highest purity available. Cloning, Overproduction, and Purification The gene (ID code PH1381) encoding the P. horikoshii IF-5A (PhoIF-5A) was amplified by PCR and placed under control of the T7 phage promoter on the expression plasmid pET-22b (19). Expression was induced with IPTG, and the resulting protein was purified to apparent homogeneity by ion-exchange chromatography on S-Sepharose. Crystallization and Data Collection The purified protein PhoIF-5A was concentrated to 10 mg ml–1 in 0.1 M Hepes-NaOH buffer (pH 7.5). Crystallization attempts were made by the hanging-drop vapor diffusion method at 18°C using crystal screen kits (Hampton Research) as reservoir solutions. Each drop was formed by mixing equal volumes (2 µl:2 µl) of protein and reservoir solution. Crystals of PhoIF-5A were obtained under conditions of 10% isopropanol and 20% PEG 4000 in one week with a size of 0.1 × 0.1 × 0.2 mm3. For data collection, the crystals of PhoIF-5A were transferred into a cryoprotectant solution containing 20% sucrose in reservoir solution, then mounted in a nylon loop and flash-frozen in a nitrogen stream at 100 K. X-ray diffraction data were collected on beamline BL41XU at SPring-8 of Japan, integrated, scaled and merged by the MOSFLM (20) and SCALA (21) programs. A crystal of PhoIF-5A diffracts to 2.0 Å and belongs to space group P32 with cell dimensions of a = b = 93.3 Å, c =39.4 Å, and γ = 120°. Crystals of PhoIF-5A contain three molecules per asymmetric unit, corresponding to a VM of 2.16 Å3 Da–1. Assuming a value of 0.74 cm3 g–1 for the protein partial specific volume (22), the calculated solvent content in the crystal is 43.1%. The statistics of data collection are summarized in Table 1. Structure Determination and Refinement The crystal structure of PhoIF-5A was determined by the molecular replacement method using the program MOLREP (23). Two search models of archaeal homologues from M. jannaschii (ID: 2EIF) and P. aerophilum (ID: 1BKB), which have 46.4 and 48% identical residues with PhoIF-5A, respectively, were constructed based on the alignment of amino acid sequences. Structure refinement was performed using the program CNS (24), and the 10% reflection data were set aside for the calculation of the free R-factor to monitor the refinement. After the first step of the refinement at 2.5 Å resolution, a model of PhoIF5A was rebuilt using the program O (25). Non-crystallographic symmetry (NCS) restraints were applied throughout the refinement. The coordinates have been deposited in the Protein Data Bank (ID: 1IZ6). RESULTS AND DISCUSSION Structure Determination and Quality In spite of the presence of three independent molecules (MolA, MolB, and MolC) in an asymmetry unit, the self-rotation function did not reveal any local symmetry. However, the native Patterson function that was subsequently calculated showed a peak at translation vector [0.67, 0.33, 0.43] (fractional coordinate), which means that at least two molecules have nearly identical orientations in the asymmetric unit. Table 2 shows the results of the molecular replacement calculated by the program MOLREP. The best solution was obtained from space group P32 and a search model of 1BKB, which gave a correlation coefficient of 40.3% and an R factor of 52.5%. Two of the three independent molecules have nearly identical orientations with a shift vector of [0.67, 0.33, 0.43]. The initial model constructed from 1BKB contained 134 residues (out of a total 136 residues) of which 62 residues possessed full side chains. After generating all side chains by computer, the first round of refinement, including rigid-body, positional, temperature and simulated annealing, was carried out at 2.5 Å resolution, which gave free R- and R-factors of 42.9 and 33.4%, respectively. The structure was remodeled using program O. After several rounds of refinement and manual fitting, the structure was refined to the free R- and R-factors of 23.7 and 19.9%, respectively, at 2.0 Å resolution. The refined structure contains 407 residues [136 (MolA), 136 (MolB), and 135 (MolC)] and 278 water molecules in the asymmetric unit. The N-terminal Met and C-terminal Gln residues are disordered, and the loop L1 (residues 33–41) has weak density. The model has geometries close to ideal with rmsd of 0.009 Å and 1.5° from ideal values for bond lengths and angles, respectively. When the structure was checked using PROCHECK (26), 91.2% of the non-glycine and non-proline residues fell in the most favored regions and 8.8% in the additional allowed region of the Ramachandran plot. The refinement statistics are summarized in Table 3. Overall Structure The refined model of PhoIF-5A is a beta-rich structure with 52.9% β-strand, 8.0% helical (including 310-helix), 18% turn, and 27.1% unclassified coil. PhoIF-5A consists of two distinct domains, an N-domain (residues 1–69) and a C-domain (residues 72–138) (Fig. 1). The secondary structure of PhoIF-5A as defined by the program DSSP (27) is shown in Fig. 2. The N-domain has an SH3-like barrel motif consisting of a 310-helix (α1) and six-stranded (β1, β2, β3, β4, β5, and β6) anti-parallel highly twisted β-sheets. The N-domain has a long hairpin loop, L1 (residues 33–41), including a specific residue, Lys37, that is supposed to be modified to hypusine residue. A quarter of the long β3 strand forms hydrogen bonds with β2, and the remaining part with β4. The β2 strand and the N-terminal portion of β3 are approximately perpendicular to strands β3, β4, β5, and β6. This highly twisted β-sheet forms a distorted β-barrel with a short 310-helix on the top. Further, hydrophobic residues Val7, Val9, Leu12, Ile18, Ile20, Ile27, Val32, Ala43, Ile45, Ile58, Val66, and Val68 form a hydrophobic core inside the β-barrel. The C-domain has an oligonucleotide/oligosaccharide binding fold (OB-fold), comprising two short α-helices (α2 and α3) and a five-stranded (β7, β8, β9, β10, and β11) anti-parallel β-barrel arranged differently from the N-terminal β-barrel. The top of C-terminal β-barrel is capped by a loop between α3 and β10. The long β7 strand forms hydrogen bonds with β8 at the center of the peptide chain. Like the N-domain, the C-terminal β-barrel also forms a hydrophobic core with residues Ala76, Val78, Ile81, Val86, Ile88, Phe97, Val99, Ile101, Val105, Leu113, Val119, and Ile130. The two domains of PhoIF-5A are connected by a peptide linker (residues 70–71), which joins the last β-strand, β6, of N-domain and the first β-strand, β7, of the C-domain. The interface of the two domains is formed by residues from β2, the loop between β4 and β5, and β10. The contact between the two domains involves several hydrogen bonds and a hydrophobic core including residues Tyr17, Pro24, Ile50, Phe51, Ile71, Trp122, Thr124, and Leu125. PhoIF-5A has a concave surface between the N- and C-domains with an approximate size of 6.5 Å × 6 Å × 15 Å calculated from the three independent molecules (MolA, MolB, and MolC) in the asymmetry unit. Structure Comparison The amino acid sequence of PhoIF-5A was aligned with those of two archaeal homologues as well as eukaryotic homologues using the program CLUSTAL W (28), as shown in Fig. 2. Most of the conserved sequences are within the N-domain, while the C-domain is less conserved. The overall structure of PhoIF-5A displays significant similarity to those of archaeal homologues (Fig. 3a). Superposition of the entire PhoIF-5A with the two homologues PaeIF-5A and MjaIF-5A using the program LSQABK (29) gave rmsd values of 1.276 and 1.690 Å, respectively, for 117 Cα atoms. When independent domains were superimposed, the N-domains of the three archaeal IF-5As have a very fixed structure (except loop L1) as shown by the small rmsd values (0.624 and 0.679 Å), whereas the rmsd values of the C-domains are 1.329 and 1.824 Å (Table 4). Distinct rmsd values for the two domains were also obtained by comparison of the three independent molecules of PhoIF-5A in the asymmetric unit, as shown in Fig. 3b and Table 4, suggesting inter-domain movement within the molecule. Thus, the peptide linker of the two domains mediates an inter-domain flexibility of about 7°. Possible RNA-Binding Site The N-domain of PhoIF-5A contains an SH3-like barrel motif, while the C-domain folds in an OB-fold. The DALI-server (30) shows that SH3-like barrel motifs are found in the DNA binding domain of HIV-integrase (31), the repressor of the E. coli biotin biosynthetic operon (32), and the C-terminal domain of the ribosomal protein L2-RBD (33). On the other hand, the OB-fold is one of the most common RNA binding modules and is found in several RNA binding proteins that are known to be associated with translation, including initiation factors, such as IF-1 (34) and eIF-2α (35), ribosomal proteins, such as S1 (36), S17 (37), and L2 (33), and the N-terminal domain of aspartyl tRNA synthetase (38). Interestingly, the ribosomal protein L2-RBD, like eIF-5A, has an SH3-like barrel motif and an OB-fold joined by a flexible peptide (33). However, the N-terminal domain of L2-RBD is an OB-fold and the C-terminal domain is an SH3-like barrel, the reverse of the situation in eIF-5A. Nevertheless, these findings strongly suggest that eIF-5A may interact with nucleic acids, particularly RNA. Indeed, it is known that eIF-5A is capable of binding to the Rev response element and U6 RNA in vitro (39). Furthermore, Xu and Chan reported that yeast eIF-5A specifically binds RNA containing the consensus sequence AAAUGUCACAC (15). It is generally known that the electrostatic interactions between exposed positively charged residues and the phosphate backbone of RNA, and stacking interactions between exposed aromatic side chains and non-base-paired RNA nucleotides are common features of protein-RNA interactions (40). As for the electrostatic potential of PhoIF-5A, the concave surface between the N- and C-domains is positively charged, as depicted in Fig. 4. Positively charged residues Lys13, Arg16, and Arg26 in the N-domain, and Lys73 and Arg132 in the C-domain are clustered at the interface of the two domains (Fig. 5). Among them, Lys13 and Lys73 are highly conserved as positive charged residues (either lysine or arginine) in five eIF-5As including yeast and human eIF-5A. In addition, two highly conserved aromatic residues, Tyr17 and Phe51, are located at the valley between the N- and C-domains. These structural features, including inter-domain flexibility, suggest that the concave surface between the N- and C-domains may present a potential surface for interaction with RNA, and that the inter-domain flexibility may become ordered upon RNA binding. It has been reported that hypusine formation is tightly coupled to cell proliferation and is essential for cell survival (6, 7, 11). Disruption of either the eIF-5A or deoxyhypusine synthase gene in yeast leads to a lethal phenotype (12–14). Further, it has been reported that this modification is required for the RNA binding activity of eIF-5A (15). Apparently, a tip of the loop L1 is positively charged, due to the Lys37 and His38 residues. Modification of Lys37 to hypusin further increases the positive charge. It is thus assumed that the tip of loop L1 may be another RNA binding site in eIF-5A, although the possibility that the modification of Lys37 could cause a structural change that results in the formation of an RNA binding site(s) in eIF-5A cannot be excluded. The unambiguous assignment of the RNA binding site must await the determination of the crystal structure of eIF-5A in complex with RNA containing the consensus sequence (15). In contrast, the molecular surface of the opposite side of the concave surface in the C-domain is highly negatively charged, as indicated in Fig. 4. It is thus unlikely that the opposite side of the concave surface of the C-domain interacts directly with RNA. It has been proposed that eIF-5A functions as a bimodular protein, capable of interacting with both protein and nucleic acid (39). Indeed, it has been reported that eIF-5A interacts with several proteins, including deoxyhypusine synthase (41), transglutaminase II (42), L5 (43), CRM1 (44), and exportin 4 (45). It is thus tempting to speculate that eIF-5A may function as a bimodular protein, with RNA binding sites at the concave surface and loop L1, and protein binding sites at the C-domain. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and by a grant from the National Project on Protein Structural and Functional Analyses. We thank Dr. M. Kawamoto of the SPring-8, Japan, for his help in data collection and Dr. Y. Kakuta for valuable comments and suggestions on the manuscript. + To whom correspondence should be addressed. Tel/Fax: +81-92-642-2853, E-mail: mkimura@agr.kyushu-u.ac.jp View largeDownload slide Fig. 1. AstereoscopicdrawingofthetranslationinitiationfactorPhoIF-5AfromP.horikoshii. The balls-and-sticks represent the specific amino acid Lys37 that is supposed to be modified to hypusine. View largeDownload slide Fig. 1. AstereoscopicdrawingofthetranslationinitiationfactorPhoIF-5AfromP.horikoshii. The balls-and-sticks represent the specific amino acid Lys37 that is supposed to be modified to hypusine. View largeDownload slide Fig. 2. Sequencecomparisonofthetranslationinitiationfactor5As. The alignment of amino acid sequences was first calculated by CLUSTAL W (28) and modified based on the secondary structures of PhoIF-5A (present work), MjaIF-5A (17), and PaeIF-5A (18). The amino acid residues are shown as follows: completely identical (pink), conserved change (yellow), completely identical only in archaeal IF-5As (light blue). The secondary structure elements are defined by the DSSP program (27). Lys37, which is modified to hypusine, is marked by an asterisk. View largeDownload slide Fig. 2. Sequencecomparisonofthetranslationinitiationfactor5As. The alignment of amino acid sequences was first calculated by CLUSTAL W (28) and modified based on the secondary structures of PhoIF-5A (present work), MjaIF-5A (17), and PaeIF-5A (18). The amino acid residues are shown as follows: completely identical (pink), conserved change (yellow), completely identical only in archaeal IF-5As (light blue). The secondary structure elements are defined by the DSSP program (27). Lys37, which is modified to hypusine, is marked by an asterisk. View largeDownload slide Fig. 3. StructuralcomparisonofthearchaealIF-5As. a: Superimposition of the backbone atoms of three archaeal homologues, PhoIF-5A (MolA, red), PaeIF-5A (green) (18), and MjaIF-5A (yellow) (17). b: Superimposition of the backbone atoms of three independent copies in the asymmetric unit of PhoIF-5A; MolA (red), MolB (green), and MolC (yellow). View largeDownload slide Fig. 3. StructuralcomparisonofthearchaealIF-5As. a: Superimposition of the backbone atoms of three archaeal homologues, PhoIF-5A (MolA, red), PaeIF-5A (green) (18), and MjaIF-5A (yellow) (17). b: Superimposition of the backbone atoms of three independent copies in the asymmetric unit of PhoIF-5A; MolA (red), MolB (green), and MolC (yellow). View largeDownload slide Fig. 4. SurfacerepresentationoftheelectrostaticpotentialofPhoIF-5A. Surface representation of the electrostatic potential of PhoIF-5A was calculated by GRASP (46). The surface potential is displayed as a color gradient from red (negative) to blue (positive). Each view is rotated by 90°. View largeDownload slide Fig. 4. SurfacerepresentationoftheelectrostaticpotentialofPhoIF-5A. Surface representation of the electrostatic potential of PhoIF-5A was calculated by GRASP (46). The surface potential is displayed as a color gradient from red (negative) to blue (positive). Each view is rotated by 90°. View largeDownload slide Fig. 5. AclusterofpositivelychargedresiduesattheputativeRNA-bindingregionofPhoIF-5A. a: The balls-and-sticks represent positive charged residues that may contribute to RNA binding. b: View rotated 90° from (a). View largeDownload slide Fig. 5. AclusterofpositivelychargedresiduesattheputativeRNA-bindingregionofPhoIF-5A. a: The balls-and-sticks represent positive charged residues that may contribute to RNA binding. b: View rotated 90° from (a). Table 1. Summary of data collection. Space group  P32  Unit cell  a = b = 93.3 Å, c = 39.2 Å, γ = 120°  Z′a  3 (VM = 2.16 Å3/Da)  Wavelength (Å)  0.90  Resolution (Å)b  100–2.0 (2.11–2.0)  Number of observed reflections  148,604  Unique reflections  25,949  Completeness (%)  100 (99.9)  Averaged redundancy  5.7 (5.7)  Averaged I/σ (I)  8.5 (2.4)  Rmeans (%)c  8.1 (34.9)  Space group  P32  Unit cell  a = b = 93.3 Å, c = 39.2 Å, γ = 120°  Z′a  3 (VM = 2.16 Å3/Da)  Wavelength (Å)  0.90  Resolution (Å)b  100–2.0 (2.11–2.0)  Number of observed reflections  148,604  Unique reflections  25,949  Completeness (%)  100 (99.9)  Averaged redundancy  5.7 (5.7)  Averaged I/σ (I)  8.5 (2.4)  Rmeans (%)c  8.1 (34.9)  aZ′ is the number of molecules in an asymmetric unit. bValues in parentheses are for the outermost resolution shell. cRmeans = Σh[m/(m – 1)]1/2Σj|<I>h – Ih,j|ΣhΣjIh,j, where <I>h is the mean intensity of symmetry-equivalent reflections and m is redundancy. View Large Table 2. Cross-rotation and translation function statistics. Model    θ1 (°)  θ2 (°)  θ3(°)  x  y  z  CC (%)  R (%)    Cross rotation function (P3x)  1BKB  answer1  22.79  67.92  –81.54        4.56      answer2  87.68  57.23  74.66        4.36      answer3  44.06  104.45  37.07        4.02      answer4  39.06  37.91  –80.41        3.73      answer5  57.39  69.98  183.05        3.72    2EIF  answer1  58.87  107.00  32.9        4.38      answer2  82.72  100.71  66.89        4.09      answer3  75.60  72.56  119.94        4.08      answer4  65.06  108.67  45.80        3.88      answer5  4.33  46.90  127.29        3.80      Translation function  1BKB  P32                    Third site using two sites of (22.79, 67.92, –81.54, 0.167, 0.118, 0.000) and (32.69, 38.81, –75.54, 0.797, 0.448, 0.334)    answer1  39.06  37.91  –80.41  0.462  0.789  0.771  40.5  52.5    answer2  39.06  37.91  –80.41  0.381  0.777  0.044  36.3  54.4    answer3  39.06  37.91  –80.41  0.477  0.688  0.815  36.1  54.0    P31                    Third site using two sites of (22.79, 67.92, –81.50, 0.607, 0.288, 0.000) and (39.06, 37.91, –80.41, 0.189, 0.239, 0.695)    answer1  22.79  67.92   –81.54  0.277  0.624  0.431  31.4  56.9    answer2  22.79  67.92  –81.54  0.218  0.144  0.036  30.3  56.7    answer3  22.79  67.92  –81.54  0.099  0.788  0.944  30.3  56.8  2EIF  P32                    Third site using two sites of (58.87, 107.00, 32.95, 0.878, 0.050, 0.000) and (82.72, 100.71, 66.89, 0.544, 0.345, 0.332)    answer1  82.72  100.71  66.89  0.203  0.674  0.758  36.9  54.6    answer2  82.72  100.71  66.89  0.207  0.672  0.649  32.7  55.8    answer3  82.72  100.71  66.89  0.194  0.643  0.874  32.6  55.6    P31                    Third site using two sites of (58.87, 107.00, 32.95, 0.377, 0.284, 0.000) and (82.72, 100.71, 66.89, 0.056, 0.362, 0.584)    answer1  32.38  50.18  –65.46  0.394  0.421  0.082  31.5  55.8    answer2  32.38  50.18  –65.46  0.713  0.471  0.874  30.7  56.2    answer3  32.38  50.18  –65.46  0.200  0.295  0.534  30.7  56.3  Model    θ1 (°)  θ2 (°)  θ3(°)  x  y  z  CC (%)  R (%)    Cross rotation function (P3x)  1BKB  answer1  22.79  67.92  –81.54        4.56      answer2  87.68  57.23  74.66        4.36      answer3  44.06  104.45  37.07        4.02      answer4  39.06  37.91  –80.41        3.73      answer5  57.39  69.98  183.05        3.72    2EIF  answer1  58.87  107.00  32.9        4.38      answer2  82.72  100.71  66.89        4.09      answer3  75.60  72.56  119.94        4.08      answer4  65.06  108.67  45.80        3.88      answer5  4.33  46.90  127.29        3.80      Translation function  1BKB  P32                    Third site using two sites of (22.79, 67.92, –81.54, 0.167, 0.118, 0.000) and (32.69, 38.81, –75.54, 0.797, 0.448, 0.334)    answer1  39.06  37.91  –80.41  0.462  0.789  0.771  40.5  52.5    answer2  39.06  37.91  –80.41  0.381  0.777  0.044  36.3  54.4    answer3  39.06  37.91  –80.41  0.477  0.688  0.815  36.1  54.0    P31                    Third site using two sites of (22.79, 67.92, –81.50, 0.607, 0.288, 0.000) and (39.06, 37.91, –80.41, 0.189, 0.239, 0.695)    answer1  22.79  67.92   –81.54  0.277  0.624  0.431  31.4  56.9    answer2  22.79  67.92  –81.54  0.218  0.144  0.036  30.3  56.7    answer3  22.79  67.92  –81.54  0.099  0.788  0.944  30.3  56.8  2EIF  P32                    Third site using two sites of (58.87, 107.00, 32.95, 0.878, 0.050, 0.000) and (82.72, 100.71, 66.89, 0.544, 0.345, 0.332)    answer1  82.72  100.71  66.89  0.203  0.674  0.758  36.9  54.6    answer2  82.72  100.71  66.89  0.207  0.672  0.649  32.7  55.8    answer3  82.72  100.71  66.89  0.194  0.643  0.874  32.6  55.6    P31                    Third site using two sites of (58.87, 107.00, 32.95, 0.377, 0.284, 0.000) and (82.72, 100.71, 66.89, 0.056, 0.362, 0.584)    answer1  32.38  50.18  –65.46  0.394  0.421  0.082  31.5  55.8    answer2  32.38  50.18  –65.46  0.713  0.471  0.874  30.7  56.2    answer3  32.38  50.18  –65.46  0.200  0.295  0.534  30.7  56.3  View Large Table 3. Final refinement statistics. Resolution range (Å)  10.0–2.0  Number of reflections  25,746  Completeness (%)  100  Total number of non-hydrogen atoms     Protein  3,158   Solvent  279  R-factor (%)a  18.5  R-free-factor (%)b  23.6  rmsd deviation from standard values     Bond lengths (Å)  0.009   Bond angles (deg)  1.628  Average B-factor (Å2)     Protein  27.7   Solvent  37.4  Ramachandran plotc     Residues in most favored regions (%)  91.2   Residues in additional allowed regions (%)  8.8  Resolution range (Å)  10.0–2.0  Number of reflections  25,746  Completeness (%)  100  Total number of non-hydrogen atoms     Protein  3,158   Solvent  279  R-factor (%)a  18.5  R-free-factor (%)b  23.6  rmsd deviation from standard values     Bond lengths (Å)  0.009   Bond angles (deg)  1.628  Average B-factor (Å2)     Protein  27.7   Solvent  37.4  Ramachandran plotc     Residues in most favored regions (%)  91.2   Residues in additional allowed regions (%)  8.8  aR-factor = Σ|Fobs – Fcal|/ΣFobs, where Fobs and Fcal are observed and calculated structure factor amplitudes. bR-free-factor value was calculated for R-factor, using only an unrefined subset of reflections data (10%). cRamachandran plot was calculated by PROCHECK (26). View Large Table 4. Superposition of IF-5As against PhoIF-5A (MolA).   rmsda    Fitting by N-domainb    Fitting by C-domainc  Fitting by alld    N-domain  C-domain    N-domain  C-domain  PhoIF-5A (MolB)  0.447  2.754    3.861  0.989  1.216  PhoIF-5A (MolC)  0.566  2.424    2.977  0.968  1.088  PaeIF-5A  0.624  2.325    3.740  1.329  1.276  MjaIF-5A  0.679  3.845    4.049  1.824  1.690    rmsda    Fitting by N-domainb    Fitting by C-domainc  Fitting by alld    N-domain  C-domain    N-domain  C-domain  PhoIF-5A (MolB)  0.447  2.754    3.861  0.989  1.216  PhoIF-5A (MolC)  0.566  2.424    2.977  0.968  1.088  PaeIF-5A  0.624  2.325    3.740  1.329  1.276  MjaIF-5A  0.679  3.845    4.049  1.824  1.690  armsd = (Σi(xi,molecule1 – xi,molecule2)2/N)1/2, xi = (xi, yi, zi). brmsd values are calculated using Cα atoms of 59 residues (without loop1) and 58 residues in N- and C-domain, respectively, after fitting by N-domain. crmsd values are calculated using Cα atoms of 59 residues (without loop1) and 58 residues in N- and C-domain, respectively, after fitting by C-domain. drmsd values are calculated using Cα atoms of 117 residues. View Large References 1. Kemper, W.M., Berry, K.W., and Merrick, W.C. ( 1976) Purification and properties of rabbit reticulocyte protein synthesis initiation factors M2Balpha and M2Bbeta. J. Biol. Chem.  251, 5551–5557 Google Scholar 2. Benne, R. and Hershey, J.W. ( 1978) The mechanism of action of protein synthesis initiation factors from rabbit reticulocytes. J. Biol. Chem.  253, 3078–3087 Google Scholar 3. Kang, H.A. and Hershey, J.W.B. ( 1994) Effect of initiation factor eIF-5A depletion on protein synthesis and proliferation of Saccharomyces cerevisiae. J. Biol. Chem.  269, 3934–3940 Google Scholar 4. Bevec, D. and Hauber, J. ( 1997) Eukaryotic initiation factor 5A activity and HIV-Rev function. Biol. Signals  6, 124–133 Google Scholar 5. Wang, T.-W., Ku, L., Wang, D., and Thompson, J.E. ( 2001) Isolation and characterization of senescence-induced cDNA encoding deoxyhypusine synthase and eukaryotic translation initiation factor 5A from tomato. J. Biol. Chem.  276, 17541–17549 Google Scholar 6. Park, M.H., Wolff, E.C., and Folk, J.E. ( 1993) Hypusine: its post-translational formation in eukaryotic initiation factor 5A and its potential role in cellular regulation. Biofactors  4, 95–104 Google Scholar 7. Chen, K.Y. and Liu, A.Y. ( 1997) Biochemistry and function of hypusine formation on eukaryotic initiation factor 5A. Biol. Signals  6, 105–109 Google Scholar 8. Park, M.H., Cooper, H.I., and Folk, J.E. ( 1982) The biosynthesis of protein-bound hypusine (N-epsilon-(4-amino-2-hydroxybutyl) lysine). Lysine as the amino acid precursor and the intermediate role of deoxyhypusine (N-epsilon-(4-aminobutyl) lysine). J. Biol. Chem.  257, 7219–7222 Google Scholar 9. Park, M.H. and Wolff, E.C. ( 1988) Cell-free synthesis of deoxyhypusine. Separation of protein substrate and enzyme and identification of 1,3-diaminopropane as a product of spermidine cleavage. J. Biol. Chem.  263, 15264–15269 Google Scholar 10. Wolff, E.C., Park, M.H., and Folk, J.E. ( 1990) Cleavage of spermidine as the first step in deoxyhypusine synthesis. The role of NAD. J. Biol. Chem.  265, 4793–4799 Google Scholar 11. Park, M.H., Lee, Y.B., and Joe, Y.A. ( 1997) Hypusine is essential for eukaryotic cell proliferation. Biol. Signals  6, 115–123 Google Scholar 12. Park, M.H., Joe, Y.A., and Kang, K.R. ( 1998) Deoxyhypusine synthase activity is essential for cell viability in the yeast Saccharomyces cerevisiae. J. Biol. Chem.  273, 1677–1683 Google Scholar 13. Schnier, J., Schwelberger, H.G., Smit-McBride, Z., Kang, H.A., and Hershey, J.W.B. ( 1991) Translation initiation factor 5A and its hypusine modification are essential for cell viability in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol.  11, 3105–3144 Google Scholar 14. Sasaki, K., Abid, M.R., and Miyazaki, M. ( 1996) Deoxyhypusine synthase gene is essential for cell viability in the yeast Saccharomyces cerevisiae. FEBS Lett.  384, 151–154 Google Scholar 15. Xu, A. and Chen, K.Y. ( 2001) Hypusine is required for a sequence-specific interaction of eukaryotic initiation factor 5A with postsystematic evolution of ligands by exponential enrichment RNA. J. Biol. Chem.  276, 2555–2561 Google Scholar 16. Kawarabayashi, Y., Sawada, M., Horikawa, H., Haikawa, Y., Hino, Y., Yamamoto, S., Sekine, M., Baba, S., Kosugi, H., Hosoyama, A., Nagai, Y., Sakai, M., Ogura, K., Otsuka, R., Nakazawa, H., Takamiya, M., Ohfuku, Y., Funahashi, T., Tanaka, T., Kudoh, Y., Yamazaki, J., Kushida, N., Oguchi, A., Aoki, K., and Kikuchi, H. ( 1998) Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3. DNA Res.  5, 55–76 Google Scholar 17. Kim, K.K., Hung, L-W., Yokota, H., Kim, R., and Kim, S-H. ( 1998) Crystal structure of eukaryotic translation initiation factor 5A from Methanococcus jannaschii at 1.8 Å resolution. Proc. Natl. Acad. Sci. USA  95, 10419–10424 Google Scholar 18. Peat, T.S., Newman, J., Waldo, G.S., Berendzen, J., and Terwilliger, T.C. ( 1998) Structure of translation initiation factor 5A from Pyrobaculum aerophilum at 1.75 Å resolution. Structure  6, 1207–1214 Google Scholar 19. Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. ( 1990) Use of T7 RNA polymerase to direct the expression of cloned genes. Methods Enzymol.  185, 60–89 Google Scholar 20. Leslie, A.G.W. ( 1993) Auto-indexing of rotation diffraction images and parameter refinement in Proceeding of the CCP4 Study Weekend (Sawyer, L., Isaacs, N., and Bailey, S., eds.) pp. 44–51, Daresbury Laboratory, Daresbury, UK Google Scholar 21. Evans, P.R. ( 1997) Scaling of MAD data in Proceeding of the CCP4 Study Weekend (Wilson, K.S., Davies, G., Ashton, A.W., and Bailey, S., eds.) pp. 97–102, Daresbury Laboratory, Daresbury, UK Google Scholar 22. Mattews, B.W. ( 1968) Solvent content of protein crystal. J. Mol. Biol.  33, 491–497 Google Scholar 23. Vagin, A. and Teplyakov, A. ( 1997) MOLREP: An automated program for molecular replacement. J. Appl. Crystallogr.  30, 1022–1025 Google Scholar 24. Brunger, A.T., Adams, P.D., Clore, G.M., Delano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jlang, J-S., Kuszewski, J., Nilges, N., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T., and Warren, G.L. ( 1998) Crystallography and NMR System (CNS): A new software system for macromolecular structure determination. Acta Crystallogr. D   54, 905–921 Google Scholar 25. Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. ( 1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A   47, 110–119 Google Scholar 26. Laskowski, R.A., MacArthur, M.W., Moss, D.S., and Thornton, J.M. ( 1993) PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr.  26, 283–291 Google Scholar 27. Kabsch, W. and Sander, C. ( 1983) Dictionary of protein secondary structures: Pattern recognition of hydrogen-bonded and geometrical features. Biopolymers  22, 2577–2637 Google Scholar 28. Thompson, J.D., Higgins, D.G., and Gibson, T.J. ( 1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res.  22, 4673–4680 Google Scholar 29. Kabsch, W. ( 1976) A solution for the best rotation to relate two sets of vectors. Acta Cryst. A  32, 922–923 Google Scholar 30. Holm, L. and Sander, C. ( 1993) Protein structure comparison by alignment of distance matrices. J. Mol. Biol.  233, 123–138 Google Scholar 31. Lodi, P.J., Ernst, J.A., Kuszewski, J., Hickman, A.B., Engelman, A., Craigie, R., Clore, G.M., and Gronenborn, A.M. ( 1995) Solution structure of the DNA binding domain of HIV-1 integrase. Biochemistry  34, 9826–9833 Google Scholar 32. Wilson, K.P., Shewchuk, L.M., Brennan, R.G., Otsuka, A.J., and Mathews, B.W. ( 1992) Escherichia coli biotin hole enzyme synthetase/bio repressor crystal structure delineates the biotin- and DNA-binding domains. Proc. Natl. Acad. Sci. USA  89, 9257–9261 Google Scholar 33. Nakagawa, A., Nakashima, T., Taniguchi, M., Hosaka, H., Kimura, M., and Tanaka, I. ( 1999) The three-dimensional structure of the RNA-binding domain of ribosomal protein L2; a protein at the peptidyl transferase center of the ribosome. EMBO J.  18, 1459–1467 Google Scholar 34. Sette, M., van Tilborg, P., Spurio, R., Kaptein, R., Paci, M., Gualerzi, C.O., and Boelens, R. ( 1997) The structure of the translational initiation factor IF1 from E. coli contains an oligomer-binding motif. EMBO J.  16, 1436–1443 Google Scholar 35. Nonato, M.C., Widom, J., and Clardy, J. ( 2002) Crystal structure of the N-terminal segment of human eukaryotic translation initiation factor 2α. J. Biol. Chem.  277, 17057–17061 Google Scholar 36. Bycroft, M., Hubbard, T.J., Proctor, M., Freund, S.M., and Murzin, A.G. ( 1997) The solution structure of the S1 RNA binding domain: a member of an ancient nucleic acid-binding fold. Cell  88, 235–242 Google Scholar 37. Golden, B.L., Hoffman, D.W., Ramakrishnan, V., and White, S.W. ( 1993) Ribosomal protein S17: characterization of the three-dimensional structure by 1H and 15N NMR. Biochemistry  32, 12812–12820 Google Scholar 38. Ruff, M., Krishnaswamy, S., Boeglin, M., Poterszman, A., Mitschler, A., Podjarny, A., Rees, B., Thierry, J.C., and Moras, D. ( 1991) Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp). Science  252, 1682–1689 Google Scholar 39. Liu, Y.P., Nemeroff, M., Yan, Y.P., and Chen, K.Y. ( 1997) Interaction of eukaryotic initiation factor 5A with the human immunodeficiency virus type 1 Rev response element RNA and U6 snRNA requires deoxyhypusine or hypusine modification. Biol. Signals  6, 166–174 Google Scholar 40. Burd, C.G. and Dreyfuss, G. ( 1994) Conserved structures and diversity of functions of RNA-binding proteins. Science  265, 615–621 Google Scholar 41. Lee, Y.B., Joe, Y.A., Wolff, E.C., Dimitriadis, E.K., and Park, M.H. ( 1999) Complex formation between deoxyhypusine synthase and its protein substrate, the eukaryotic translation initiation factor 5A (eIF5A) precursor. Biochem. J.  340, 273–281 Google Scholar 42. Singh, U.S., Li, Q., and Cerione, R. ( 1998) Identification of the eukaryotic initiation factor 5A as a retinoic acid-stimulated cellular binding partner for tissue transglutaminase II. J. Biol. Chem.  273, 1946–1950 Google Scholar 43. Schatz, O., Oft, M., Dascher, C., Schebesta, M., Rosorius, O., Jaksche, H., Dobrovnik, M., Bevec, D., and Hauber, J. ( 1998) Interaction of the HIV-1 Rev cofactor eukaryotic initiation factor 5A with ribosomal protein L5. Proc. Natl. Acad. Sci. USA  95, 1607–1612 Google Scholar 44. Elfgang, C., Rosorius, O., Hofer, L., Jaksche, H., Hauber, J., and Bevec, D. ( 1999) Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals. Proc. Natl. Acad. Sci. USA  96, 6229–6234 Google Scholar 45. Lipowsky, G., Bischoff, F.R., Schwarzmaier, P., Kraft, R., Kostka, S., Hartmann, E., Kutay, U., and Gorlich, D. ( 2000) Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes. EMBO J.  19, 4362–4371 Google Scholar 46. Nicholls, A., Sharp, K., and Honig, B. ( 1991) Protein folding and association; insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struct. Funct. Genet.  11, 281–296 Google Scholar Author notes 1Division of Biological Science, Graduate School of Science, Hokkaido University, Sapporo 066-0810; and 2Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581

Journal

The Journal of Biochemistry – Oxford University Press

Published: Jan 1, 2003

There are no references for this article.