Structural Features of the eIF-5A Precursor Required for Posttranslational Synthesis of Deoxyhypusine*

Eukaryotic translation initiation factor 5A (eIF-5A, older nomenclature, eIF-4D) is a highly conserved protein that contains the unusual amino acid hypusine (W-(4-amino-2-hydroxybuty1)lysine). The biosynthesis of hy- pusine occurs posttranslationally in only this protein by modification of a single lysine residue (Lyssa in the human eIF-5A precursor). The basis for the specificity of this modification with respect to the substrate protein was investigated using fragments of eIF-5A precursor protein, each containing this lysine residue, as substrates for deoxyhypusine synthase, the first enzyme in hypusine synthesis. Proteolytic fragments (5-6 kDa) of ec-eIF-5A (the precursor form of eIF-5A produced in Escherichia coli by expression of the human eIF-5A cDNA) generated by specific cleavage by endoproteinases Arg-C, Asp-N, or Glu-C, did not act as substrates for deoxyhypusine synthesis. A series of truncated forms of the eIF-5A precursor protein generated by expression in E. coli of recombinant deletion constructs from the hu- man eIF-5A cDNA were tested. Truncation of up to 9 amino acid residues (Metl-Thfl) from the NH, terminus or 64 amino acid residues (Leu’l-Ly~’~) from the COOH terminus did not significantly decrease the substrate reactivity,

Eukaryotic translation initiation factor 5A (eIF-5A, older nomenclature, eIF-4D) is a highly conserved protein that contains the unusual amino acid hypusine (W-(4-amino-2-hydroxybuty1)lysine). The biosynthesis of hypusine occurs posttranslationally in only this protein by modification of a single lysine residue (Lyssa in the human eIF-5A precursor). The basis for the specificity of this modification with respect to the substrate protein was investigated using fragments of eIF-5A precursor protein, each containing this lysine residue, as substrates for deoxyhypusine synthase, the first enzyme in hypusine synthesis. Proteolytic fragments (5-6 kDa) of ec-eIF-5A (the precursor form of eIF-5A produced in Escherichia coli by expression of the human eIF-5A cDNA) generated by specific cleavage by endoproteinases Arg-C, Asp-N, or Glu-C, did not act as substrates for deoxyhypusine synthesis. A series of truncated forms of the eIF-5A precursor protein generated by expression in E. coli of recombinant deletion constructs from the human eIF-5A cDNA were tested. Truncation of up to 9 amino acid residues (Metl-Thfl) from the NH, terminus or 64 amino acid residues (Leu'l-Ly~'~) from the COOH terminus did not significantly decrease the substrate reactivity, but removal of an additional 10 amino acids from either side did. Deletion of 34 amino acid residues (Met'-Ly~~~) from the NH, terminus or of 84 amino acid residues (A~p"-Lys~~) from the carboxyl terminus caused complete loss of substrate property. The results obtained thus far define the minimum domain of the eIF-5A precursor protein required for enzymatic deoxyhypusine synthesis as Phe30-Asp80, which corresponds to a region of high amino acid conservation in this protein throughout the eukaryotic kingdom.
Eukaryotic translation initiation factor 5A (eIF-5A, old nomenclature, eIF-4D)' is a small acidic protein that has been * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 [1][2][3]. In earlier studies eIF-5A was termed The nomenclature for initiation factors has been revised (IUB-NC eIF-4D. The abbreviations used are: eIF-5A, eukaryotic initiation factor 5A, ec-eIF-5A, the unmodified precursor form of eIF-5A, containing Lys in place of hypusine, that is produced in E. coli by overexpression of human eIF-5A cDNA 5A(nl-n2) designates peptides derived from ec-eIF-5A in which the numbers give the positions of the first and the last amino acids. When the amino acid residue at the number n l is not methionine, methionine was introduced as the NH, terminus of the peptide as indicated by "5A(Met nl-n2)." PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Arg-C, endoproteinase from mouse submaxillary gland that cleaves the peptide bond at the carboxyl side of Arg residue; Asp-N, endoproteinase from Pseudomonas suggested to participate in the first peptide bond formation during protein synthesis (1). Biogenesis of mature eIF-5A involves a unique posttranslational modification of one specific lysine residue (Lys5' in the case of the human eIF-SA precursor) to an unusual amino acid, hypusine (N"(4-amino-2-hydroxybutylllysine), which occurs exclusively in this protein (see Ref. 2 for a recent review). The biosynthesis of hypusine in a single cellular protein, the eIF-SA precursor, and of diphthamide in the eukaryotic elongation factor 2 (eEF-2) precursor, represent the two most specific protein modifications known to date (3). Hypusine formation occurs by way of two enzyme-catalyzed steps (4): (i) transfer of the butylamine moiety of the polyamine spermidine to the €-amino group of one specific lysyl residue of the eIF-5A precursor protein to form a n intermediate deoxyhypusine (N"(4-aminobuty1)lysine) (5)(6)(7) and (ii) hydroxylation of the deoxyhypusyl residue to form hypusine (8).
Hypusine and eIF-5A occur in all eukaryotic species examined (2). The hypusine synthesis rate and/or hypusine content appear to correlate with cellular proliferation in various mammalian cells, suggesting an important role for this protein in cell metabolism (2). Hypusine was shown to be essential for the activity of eIF-SA (9, 10) in stimulating methionyl puromycin synthesis, an in vitro model assay for translation initiation.
Although the precise physiological function of eIF-SA in eukaryotic cells is unknown, recent studies that involved the inactivation of the two eIF-SA genes in Saccharomyces cerevisiae provide strong evidence that eIF-SA and hypusine are vital for yeast growth (11,12). The arrest of growth of mammalian cells by agents that cause the reduction of cellular hypusine, either by depletion of spermidine (13) or by inhibition of deoxyhypusine synthase (141,' further supports a crucial role of hypusine in eukaryotes (15). eIF-SA is a highly conserved protein (11,(16)(17)(18)(19)(20)(21). There is a 99% amino acid identity between rabbit and human eIF-SA and 60-65% identity between human and yeast eIF-5A sequences (11,17). Even in the regions of nonidentity, amino acid replacements are often conservative. Sequence similarity is high in the amino-terminal half of the protein and especially in the vicinity of the hypusyl residue. The sequence of 12 amino acids surrounding the Lys residue (Lys*) that undergoes modification to hypusine, -Ser-Thr-Ser-Lys-Thr-Gly-Lys*-His-Gly-His-Ala-Lys-, is invariable in all eukaryotes. It suggests the importance of this region for a fundamental cellular function throughout eukaryotic evolution and/or for recognition of the precursor lysyl residue by the enzymes that catalyze the formation of hy-   (10)(11)(12)(13)(14)(15)(16), the numbers in parentheses indicate the amino acid residue numbers of the eIF-SA precursor to which the coding, or the complementary sequence, of the primer corresponds. The NdeI sites for the forward primers 1-6 are underlined, and the BarnHI sites for the reverse primers are shown in bold letters. cellular protein, together with the high conservation of amino acids that surround this residue, suggests a narrow specificity of deoxyhypusine synthase toward its protein substrate. In preliminary experiments, two synthetic peptides that correspond to the amino acid sequence surrounding Lys5" in the human eIF-5A precursor, a nanopeptide KTGK' OHGHAK and a hexadecamer IWMSTSKTGKSoHGHAK, were found neither to act as substrates for deoxyhypusine synthase3 nor to inhibit the synthesis of deoxyhypusine in the intact substrate protein ec-eIF-5A. In an effort to explore the structural features of the eIF-5A precursor that confer specific recognition by deoxyhypusine synthase, we carried out a systematic study on the structure-substrate function relationships of deoxyhypusine synthesis. Evidence is presented that a substantial portion of the primary structure of the substrate protein is required for recognition and modification by deoxyhypusine synthase.
Proteolytic Digestion-The eIF-5A precursor protein, ec-eIF-BA, and its truncated fragments were digested with one of the three endoproteinases, Arg-C or Asp-N in 100 nm Tris-C1, pH 8.5, or Glu-C in 100 m M Tris-C1, pH 7.8, buffer as described in the legend of Fig. 1.
Construction of Deletion Recombinant Subclones of Human eZF-5A cDNA-A series of different size constructs of human eIF-5A cDNA was prepared by PCR amplification from the full-length human eIF-5A cDNA (17) using synthetic oligonucleotide primers (Table I). These constructs were designed for insertion into PET l l a expression vector (Novagen). The PCR primers were synthesized with an extension of 16 nucleotides containing an NdeI site (CATATG) at the 5' end or a termination codon TGA followed by BamHI site (TCCTGA) at the 3' end, which is flanked by the desired coding or complementary sequences ( Table I). The conditions for PCR were as follows: 94°C for 5 min, denaturation at 94 "C for 1 min, annealing at 55 "C for 1 min, extension reaction at 74 "C for 2 min for 35 cycles, and the final extension reaction at 74 "C for 6 min. Insertion of the NdeI site at the 5' end adds an additional methionine residue at the amino terminus of the clones except those with methionine as a NH,-terminal residue. The PCR products were cleaved with NdeI and BamHI and ligated to the insertion E. C. Wolff  Deoxyhypusine Synthase Assay-The deoxyhypusine synthase assay is based on the incorporation of radioactivity from the aminobutyl side of [1,8-3H]spermidine into deoxyhypusine in the substrate protein eIF-5A precursor or its fragments. The enzyme assay was camed out according to the published method (7) with modification. A typical reaction mixture contained 0.2 M glycine.NaOH buffer, pH 9.4, 1 m M dithiothreitol, 0.5 m M NAD+, 2 pCi of [1,8-3H]spermidine (15 Ci/mmol), 6 1 5 units of deoxyhypusine synthase from rat testis, and varying amounts of substrate protein, ec-eIF-5A, or its peptide fragments, either in a purified form or as the crude lysate of the E. coli cells that expressed them. The assay mixture was incubated at 37 "C for 2 h. The radiolabeled product protein or peptides were separated by SDS-PAGE on Tricine gel (16% in acrylamide, Novex, San Diego, CA) and visualized by fluorography. For quantitation of the reaction product, the assay mixtures were precipitated with trichloroacetic acid, the trichloroacetic acid precipitates were hydrolyzed in 6 N HCl, and the radiolabeled deoxyhypusine was measured after its separation by ion exchange chromatography as described previously (7,22).

RESULTS
Because attempts to achieve deoxyhypusine synthesis using small synthetic peptides containing the sequence of amino acids surrounding Lyssa in human eIF-5A precursor were unsuccessful,3 we turned to larger peptide fragments generated by specific proteolytic cleavage of ec-eIF-SA. The three endoproteinases, Arg-C, Glu-C, and Asp-N, which catalyze cleavages at the carboxyl side of Arg, at the carboxyl side of Glu, and at the amino side of Asp, respectively, were chosen on the basis of  6 ) from a partial digest of ec-eIF-5A (1 mg/ml) with Asp-N (0.012 mg/ml, 18 h, 37 "C) was obtained after reverse phase high performance liquid chromatography on a pBondapack C,, column. The 5-kDa peptide containing Lys"" (lanes 7 and 8 ) was obtained by digestion of the ec-eIF-5A fragment 5A(20-90) (1 mg/ml) with Glu-C (10 pg/ml, 18 h, room temperature). The 6-kDa Lys"'-containing peptide (lanes 9 and 10) was generated by digestion of the recombinant deletion peptide 5A(Met10-90) (1 mg/ml) with Arg-C (IO pg/ml, 5 h, room temperature). The digests of 5A(Met10-90) and 5A(20-90) were treated with a protease inhibitor mixture (1 mhl o-phenanthroline, 1 pg/ml aprotinin, 0.5 pg/ml leupeptin) before the deoxyhypusine synthase assay. SDS-PAGE of ec-eIF-5A and its digests by Arg-C, Asp-N, and Glu-C was conducted on a precast Tricine gel (16% in acrylamide, Novex). Prior to application, the samples were heated for 2 min in buffer (62.5 mM Tris-C1, pH 6.8, 2% SDS, 10% glycerol, 1% dithiothreitol). their specificity and because there are no preferred cleavage sites for these enzymes in the vicinity of Lys5" in ec-eIF-5A. The pattern of proteolytic peptides produced vaned with the amount of enzyme and the length of digestion. None of the peptides (3-6 kDa) generated by digestion with Arg-C, Asp-N, or Glu-C served as substrates for deoxyhypusine synthesis (data not shown), with the exception of those 8-17-kDa peptides in the partial digest with Asp-N. Fig. 1 shows the Coomassie Blue-stained pattern and the fluorogram of gels of deoxyhypusine synthase reaction mixtures containing ec-eIF-SA or its Lyssa-containing peptides. Only in the 8-17-kDa peptide of the Asp-N partial digest (Fig. 1, lunes 5 and 6) was strong radiolabeling observed. No labeling was seen with other proteolytic peptides. Intact ec-eIF-5A was included in the reaction mixtures of the even numbered lanes to monitor the reaction. In each case ec-eIF-5A was effectively labeled, thus indicating that the reaction mixtures containing protease inhibitors or possibly the residual protease did not interfere with deoxyhypusine synthesis.
In an effort to define more precisely that portion of the ec-eIF-SA molecule required for the enzymatic formation of deoxyhypusine in the modification reaction, systematic deletions from the NH, terminus, from the COOH terminus, or from both were carried out by deletion recombinant subcloning of human eIF-5A cDNA, and the resulting fragments were tested as deoxyhypusine synthase substrates (Figs. 2 and 3). Most transformants expressed the desired peptides. However, the level of expression varied with different deletion subclones and transformants. In general, the levels of recombinant peptides of low molecular mass (<6 kDa) in the transformants were low, presumably due to the rapid degradation of small peptides by E.
coli. Peptides representing stepwise deletion from the COOH terminus of ec-eIF-5A, 5A(1-154), 5A(1-120), 5A(1-90), and 5A(1-80) (Fig. 2) were efficiently labeled when lysates of the transformants expressing them were tested in the deoxyhy-pusine synthase reaction. Although, in general, efficiency as a substrate declined progressively with deletions from the COOH terminus (Table 11, Fig. 2), it appears that the carboxyl half (Val"-Ly~'~~) of ec-eIF-5A is not an absolute requirement for deoxyhypusine synthesis. No radiolabeling was detected in peptides 5A(1-60) and 5A(1-70), indicating that the boundary of the minimum interaction domain on the carboxyl side of ec-eIF-5A lies between Glu7' and Asp". In order to assess the requirement for the amino acid sequence on the NH, side of Lys"", the effects of stepwise deletion from the NH, terminus were examined (Fig. 2). Deletion of 9 amino acids from the NH, terminus caused only a small reduction in substrate efficiency (Fig. 2, compare 1-90 and 10-90). Deletion of 19 or 29 NH,terminal amino acids, however, decreased significantly the substrate property (Table 11, Fig. 2). Further deletions from the NH, terminus, as in the cases of 5A(Met35-154), and 5A(43-154) totally abolished the substrate capacity. These findings, together with the COOH-terminal deletion studies, establish Phe3'-AspR0 as the minimum domain of ec-eIF-5A required for deoxyhypusine synthesis. Indeed, deoxyhypusine synthesis in the peptide fragment with this sequence, 5A(Met30-80), even when its expression level was low in the transformant, was clearly demonstrable (Fig. 2). The schematic diagram presented in Fig. 3 illustrates the relationship between the substrate potential of the peptides and the conservation of amino acid sequence of eIF-BAS from several eukaryotes, including human (17), alfalfa (18), tobacco (191, slime mold (201, yeast (111, and chick embryo (21). DISCUSSION Among the many amino acids that are derived by posttranslational modification, hypusine is a most remarkable one, vital for eukaryotic cell proliferation, yet occurring at a single position in only one cellular protein. In order for deoxyhypusine synthase, the first enzyme in hypusine biosynthesis, to act on a

B T A I~ I1
Efficiency of deoxyhypusine synthesis in the eIF-5A precursor and its peptide fragments Each substrate (50 pmol) purified from an overproducing transformant was incubated with rat testis deoxyhypusine synthase as described under "Experimental Procedures." The amount of product formed was determined by measuring the radioactivity in deoxyhypusine after ion exchange chromatographic separation of the hydrolysed protein. single lysyl residue and exclude the multitude of other lysyl residues in the whole cell, it must exhibit an extremely narrow specificity toward its protein substrate. The present study provides evidence that a relatively large portion of the primary structure of ec-eIF-5A is required for recognition and modification by deoxyhypusine synthase and offers insights into the interaction between the protein substrate, ec-eIF-5A, and the enzyme.
The recombinant deletion studies define the minimal domain required for deoxyhypusine synthesis as Phe"'-Aspnn. This core domain, however, is far less efficient a s a substrate than the intact protein. The amino-terminal 9 amino acids (Met'-Thr') and the carboxyl-terminal portion (Leu9l-LysI5') of ec-eIF-5A do not appear to be important for the modification. The stepwise deletion of interim sequences, Gly"-Gly2' and ValR'-Gln"', however, caused significant progressive reduction in substrate efficiency. No proteolytic or deletion peptide functioned a s a better substrate than the intact protein ec-eIF-5A.
Although the results obtained with the proteolytic peptides of ec-eIF-5A provided preliminary evidence for the domain involved in deoxyhypusine synthesis, the lack of strict specificity of cleavage by the endoproteinases, Asp-N, Arg-C, and Glu-C, which hampered the purification and identification of individual cleavage products, left some doubts as to the exact primary structural requirements. However, the three unreactive peptides containing Lys"', a 6-kDa Asp-N peptide, a 5-kDa Glu-C peptide, and a 6-kDa Arg-C peptide (Fig. lA, arrows) and the reactive 8-kDa Asp-N peptide (Fig. 1, A and B, arrows) were presumed to be Asp1L-Ile61, Met4"-AspXX, Pro"-Ar$' and Asp"-Met7', respectively, based on the known specificity of the proteases, the ion exchange chromatographic properties and mobilities of the proteolytic peptides on SDS-PAGE. With this assumption, the observed substrate reactivities of these proteolytic peptides are consistent with those of the recombinant deletion peptides.
The requirement for a large portion of the primary structure of ec-eIF-5A for its modification suggests that substrate binding involves elements of secondary or tertiary structure. The domain strictly required for deoxyhypusine synthesis, Phe"-Asp"", is very basic. The sequence in the close vicinity of Lys"', Gl~'~-His"~, is especially basic and hydrophilic and is presumably exposed on the surface of the protein. This hypusine site sequence is surrounded by stretches of hydrophobic regions (Gly"-Val") and (Leu"'-Phe'"). The amino-terminal sequence (Met'-Thr') and the carboxyl-terminal portion (Leu9'-Lys'"), which are not critical elements for deoxyhypusine synthesis, are rich in acidic amino acids. Very little is known about the structural organization of eIF-5A or its precursor. Based on the amino acid sequence of ec-eIF-5A, computer-generated predictions were made on the flexibility and the secondary structure of the protein (171.' According to these analyses, the protein possesses organized NH,-terminal and COOH-terminal regions, but the middle region consists of p-turns and/or unordered residues. The site of hypusine modification resides in a flexible area flanked by inflexible hydrophobic regions (17)."All three regions appear to be essential for function as a substrate.
Upon comparison of amino acid sequences of eIF-SA'S from various species (Fig. 3 B ) it is manifest that the core domain PheR'-AspR' needed for deoxyhypusine synthesis lies in a remarkably conserved region of the protein. On the other hand, the amino acid conservation is relatively low in the regions (Met1-Thr9, Leu9'-Lys1'') not critical for deoxyhypusine synthesis. The high conservation of the domain surrounding Lys"' may have been dictated mainly by the structural requirement for its cellular function. However, on the basis of our results, it is tempting to speculate that the narrow specificity of the modification reaction also contributed to the amino acid sequence conservation of eIF-5A.
A. Abbruzzese, personal communication. Despite the well established essential role of hypusine and eIF-5A in eukaryotic cell proliferation, the specific action of eIF-5A in cell metabolism remains an open question. Although its role in general protein synthesis has been challenged (23), its possible function as an initiation factor selective for certain specific messages has not been excluded (2,231. Recent studies suggest that eIF-5A is the host cellular factor required for the Rev function essential for the human immunodeficiency virus type 1 replication (24) and open a new possibility for the role of this putative initiation factor as a posttranscriptional regulator of specific gene expression. The current results provide a foundation for future studies directed toward determination of the structural features of eIF-5A required for its physiological role in eukaryotic cells.