Crystal structure of elongation factor P from Thermus thermophilus HB8

Kyoko Hanawa-Suetsugu*, Shun-ichi Sekine†, Hiroaki Sakai*, Chie Hori-Takemoto*, Takaho Terada*†, Satoru Unzai*‡, Jeremy R. H. Tame*‡, Seiki Kuramitsu†§, Mikako Shirouzu*†, and Shigeyuki Yokoyama*†¶ʈ

*RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; †RIKEN Harima Institute at SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan; ‡Protein Design Laboratory, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; §Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan; and ¶Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Edited by Paul R. Schimmel, The Scripps Research Institute, La Jolla, CA, and approved May 13, 2004 (received for review December 26, 2003) Translation elongation factor P (EF-P) stimulates ribosomal pepti- other hand, hypusine is not found in bacteria (13). The structures dyltransferase activity. EF-P is conserved in bacteria and is essential of eIF-5A from three Archaea, Methanococcus jannaschii, Py- for cell viability. Eukarya and Archaea have an EF-P homologue, robaculum aerophilum, and Pyrococcus horikoshii, have two eukaryotic initiation factor 5A (eIF-5A). In the present study, we domains, which are composed of several ␤-strands (14–16). determined the crystal structure of EF-P from Thermus thermophi- Crystallizations of EF-P from E. coli and Aquifex aeolicus have lus HB8 at a 1.65-Å resolution. EF-P consists of three ␤-barrel been reported (7, 17). In this article, we report the crystal domains (I, II, and III), whereas eIF-5A has only two domains (N and structure of EF-P from Thermus thermophilus HB8 at a 1.65-Å C domains). Domain I of EF-P is topologically the same as the N resolution. The EF-P structures are ␤-rich and is divided into domain of eIF-5A. On the other hand, EF-P domains II and III share three ␤-barrel domains (domains I, II, and III). Domains II and the same topology as that of the eIF-5A C domain, indicating that III of EF-P share a very similar topology. The structures of domains II and III arose by duplication. Intriguingly, the N-terminal domains I and II of EF-P are superposable on the structures of half of domain II and the C-terminal half of domain III of EF-P have the M. jannaschii, P. aerophilum, and P. horikoshii eIF-5A sequence homologies to the N- and C-terminal halves, respectively, proteins. The overall structure of EF-P is strikingly similar to the

of the eIF-5A C domain. The three domains of EF-P are arranged in L-shaped structure of tRNA. BIOCHEMISTRY an ‘‘L’’ shape, with 65- and 53-Å-long arms at an angle of 95°, which is reminiscent of tRNA. Furthermore, most of the EF-P protein Materials and Methods surface is negatively charged. Therefore, EF-P mimics the tRNA Protein Preparation and Crystallization. The DNA fragment encod- shape but uses domain topologies different from those of the ing EF-P, the protein TT0860 (DNA Data Base in Japan, known tRNA-mimicry translation factors. Domain I of EF-P has a accession no. AB103477), was isolated from the T. thermophilus conserved positive charge at its tip, like the eIF-5A N domain. HB8 genome and was cloned into the expression plasmid, pET11a (Novagen). E. coli BL21(DE3) was transformed with the ranslation elongation factor P (EF-P) was found as a protein vector, and T. thermophilus EF-P was overexpressed. The protein Tthat stimulates the peptidyltransferase activity of the 70S was purified by successive chromatography steps on Q Sepharose ribosome in Escherichia coli (1). EF-P enhances dipeptide and HiLoad Superdex 75 columns (Amersham Biosciences). synthesis with N-formylmethionyl-tRNA and puromycin in vitro, Hampton Research Crystal Screen (18) was used to determine suggesting its involvement in the formation of the first peptide the initial crystallization conditions for EF-P. The final crystal- bond of a protein (2). E. coli EF-P is encoded by the efp gene and lization conditions, 100 mM Hepes-Na buffer (pH 7.6) and 1.35 consists of 188 amino acid residues (3). The efp genes are M lithium sulfate at 16°C yielded high-quality crystals suitable universally conserved in Bacteria (4). Gene interruption exper- for x-ray diffraction data collection. They belong to the space ϭ ϭ iments in E. coli revealed that the efp gene is essential for cell group P212121, with unit cell dimensions a 55.8, b 78.4, and viability and is required for protein synthesis (3). The amount of c ϭ 138.9 Å. The crystallographic asymmetric unit contains two Ϸ ͞ EF-P in E. coli cells is 1 10th of that of EF-G; 800–900 nearly identical EF-P monomers. molecules of EF-P exist in a cell, an amount consistent with 1 EF-P per 10 ribosomes (5). Data Collection and Structure Determination. The crystal structure EF-P reportedly binds to both the 30S and 50S ribosomal of EF-P was solved by the multiple isomorphous replacement subunits (6). Antibiotic sensitivity and footprinting studies have method. Three heavy-atom derivatives were prepared by soak- indicated that EF-P binds near the streptomycin-binding site of ing the EF-P crystals for 12 h in reservoir solutions containing the 16S rRNA in the 30S subunit (6). EF-P also interacts with 2 mM potassium tetrachloroaurate (III), 2 mM sodium ethyl- domains II and V of the 23S rRNA, i.e., near the peptidyltrans- mercurithiosalicylate, and 2 mM mersalyl acid, respectively ferase center (PTC) (6, 7). Ribosome reconstitution experiments (Table 1). All the native and heavy-atom derivative data sets have shown that the L16 ribosomal protein or its N-terminal were collected from frozen crystals at 90 K by using synchro- 47-residue fragment was required for EF-P-mediated peptide tron radiation at the SPring-8 beam lines (Hyogo, Japan). The bond synthesis, whereas L11, L15, or L7͞L12 were not (8–10). data were processed with the program HKL2000 (19). Deter- Eukarya and Archaea seem to lack EF-P, although a similar mination of the heavy atom positions and calculation of the function may be mediated by eukaryotic initiation factor 5A (eIF-5A) (4, 6). The eIF-5A protein is composed of Ϸ140 amino Ϸ acid residues and is shorter than EF-P by 40 residues. Com- This paper was submitted directly (Track II) to the PNAS office. plete intracellular depletion of eIF-5A results in cell growth Abbreviations: EF, elongation factor; eIF-5A, eukaryotic initiation factor 5A; rmsd, rms inhibition; however, protein synthesis seems to be only slightly deviation. reduced (11). eIF-5A is a unique cellular protein that contains Data deposition: The atomic coordinates and structure factors have been deposited in the ␧ the unusual amino acid hypusine [N -(4-aminobutyl-2-hydroxy)- Protein Data Bank, www.pdb.org (PDB ID code 1UEB). L-lysine], which is formed by posttranslational modification of a ʈTo whom correspondence should be addressed. E-mail: [email protected]. specific lysine residue. The enzyme that modifies lysine to ac.jp. hypusine in eIF-5A is essential for yeast viability (12). On the © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308667101 PNAS ͉ June 29, 2004 ͉ vol. 101 ͉ no. 26 ͉ 9595–9600 Downloaded by guest on October 1, 2021 Table 1. Crystallographic data

Native K2AuCl4 EMTS Mersaryl acid

Data collection Resolution,* Å 50–1.65 (1.71–4.65) 50–2.37 (2.45–2.37) 50–2.11 (2.19–2.11) 50–2.11 (2.19–2.11) Observed reflections, n 451,416 103,399 133,338 156,921 Unique reflections, n 62,691 21,379 30,131 30,123 Completeness,† % 99.9 (100.0) 98.8 (92.6) 99.9 (99.8) 99.4 (98.8) ‡ Rsym, % 7.7 (49.6) 3.4 (9.1) 5.5 (14.6) 8.1 (26.9) I͞␴(I)§ 26.4 (3.0) 44.9 (22.1) 26.4 (7.9) 18.9 (5.9) Heavy atom refinement and phasing Heavy atom sites, n 111 ¶ Riso, % 6.5 15.0 24.9 Phasing power (centric–acentric)࿣ 0.58–0.75 0.42–0.50 0.33–0.34

Rcullis** 0.69 0.73 0.80 Mean FOM†† 0.46 Refinement Resolution, Å 40–1.65 Reflections, n 62,587 ‡‡ Rcryst, % 21.3 ‡‡ Rfree, % 24.1 Protein atoms, n 2797 Water atoms, n 407 rmsd bonds, Å 0.005 rmsd angles, ° 1.2 rmsd improper angles, ° 0.83

EMTS, sodium ethylmecurithiosalicylate. *Resolution range of the highest shell is listed in parentheses. †Completeness in the highest-resolution shell is listed in parentheses. ‡ Rsym ϭ͚͉Iobs Ϫ͗I͉͚͘͞Iobs, where Iobs is the observed intensity of reflection. Rsym in the highest-resolution shell is listed in parentheses. §I͞␴(I) in the highest-resolution shell is listed in parentheses. ¶ Riso ϭ͚͉Fder Ϫ Fnat͉͚͞Fnat, where Fnat and Fder are the native and derivative structure factor amplitude, respectively. ࿣ ϭ ͚ 2 ͚͞ Ϫ 2 1/2 Phasing power ( FH (FPH(obs) FPH(calc)) ) , where FH represents the calculated heavy atom structure factor amplitude. **Rcullis ϭ͚ʈFPH Ϯ FP͉ Ϫ FH͉͚͉͞FPH Ϯ FP͉. †† Mean figure of merit (FOM) ϭ͗Fbest͞F͘. ‡‡ Rcryst,free ϭ͚͉Fobs Ϫ Fcalc͉͚͞Fobs, where the crystallographic R factor is calculated including and excluding refinement reflections. In each refinement, free reflections consist of 5% of the total number of reflections.

multiple isomorphous replacement phases were carried out by and by analytical ultracentrifugation (Optima XL-1, Beckman using the program SOLVE (20). The experimental phases were Coulter). Light scattering was performed at 20°Cin20mM improved by using the RESOLVE program (20) and further Tris⅐HCl buffer (pH 7.5) containing 150 mM NaCl and 1 mM refined by using the ARP͞WARP program (21) to 1.65 Å. The DTT. Analytical ultracentrifugation was performed at 20°Cin20 improved electron density map was of high quality, which mM Tris⅐HCl buffer (pH 7.5) containing 150 mM NaCl and 5 allowed the ARP͞WARP program to automatically build an mM 2-mercaptoethanol. almost complete model of one of the EF-P monomers (mol- ecule A) in the asymmetric unit. Because the structure of the Results and Discussion other EF-P molecule in the asymmetric unit (molecule B) is Overall Structure. In the present study, we determined the crystal practically identical with that of molecule A, the molecule B structure of EF-P from T. thermophilus at a 1.65-Å resolution by model was readily produced by fitting the molecule A model the multiple isomorphous replacement method. The crystallo- to the electron density. The models were manually adjusted to graphic data are summarized in Table 1. The atomic coordinates the electron density by using the O program (22). Because no have been deposited in the Protein Data Bank (PDB ID code clear electron density was observed for the loop region (amino 1UEB). In the crystal, two molecules (A and B) are in the acid residues 139–145) of molecule B, these residues were asymmetric unit (Fig. 1A). The EF-P protein is a ␤-rich protein excluded from the coordinates. The refinement was carried out containing 16 ␤-strands and is made up of three ␤-barrel with several rounds of conventional molecular dynamics pro- domains (domains I, II, and III) (Fig. 1B). tocols with the CNS program (23), with all data in the resolution The ␤-strands of molecule A are designated as ␤1–␤16, range of 40–1.65 Å. The refinement converged to an R factor whereas the corresponding ␤-strands of molecule B are ␤1Ј– ϭ ␤ Ј of 21.3% (Rfree 24.9%) at a 1.65-Å resolution (Table 1). The 16 , to discriminate between the two molecules. In the protein final model has 91.8% and 8.2% of the amino acid residues in crystal, the ␤3-strand forms an antiparallel ␤-sheet with the the most favored and additional allowed regions, respectively, ␤3Ј-strand of the other monomer (Fig. 1A). This interaction of the Ramachandran plot, as indicated by the program connects the two monomers in the asymmetric unit, to form the PROCHECK (24). Graphic figures were created with the pro- dimer. A large, flat, six-stranded ␤-sheet is formed by ␤3, ␤4, ␤5, grams MOLSCRIPT (25) and RASTER3D (26) or GRASP (27). ␤3Ј, ␤4Ј, and ␤5Ј. The two monomers are related by a pseudo 2-fold axis, which is perpendicular to the ␤-sheet and passes near Determination of Molecular Weight in Solution. The molecular the carbonyl oxygen of His-27 on ␤3 (or ␤3Ј). The buried surface weight of T. thermophilus EF-P in solution was estimated by light area between the EF-Ps was 539 Å2, which is Ϸ5% of the scattering (DynaPro 99, Protein Solutions, Charlottesville, VA) monomer surface of EF-P. The T. thermophilus EF-P exists as a

9596 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308667101 Hanawa-Suetsugu et al. Downloaded by guest on October 1, 2021 Fig. 1. The structure of T. thermophilus EF-P. (A) Ribbon diagram showing the two molecules (A and B) in the asymmetric unit. The arrows represent ␤-strands. The three domains of each molecule are colored green, red, and blue, in dark and light tones for molecules A and B, respectively. The pseudosymmetry axisis marked in magenta. (B) Topology diagram of the T. thermophilus EF-P structure. The arrows represent ␤-strands and the ellipses represent 310-helixes. Domains I, II, and III are colored green, red, and blue, respectively. (C) Ribbon presentation of the T. thermophilus EF-P (molecule A) crystal structure (stereoview). Color coding is as in B.

monomer (20,224 Da) under physiological conditions, as shown of the EF-P molecule are similar to those of tRNA molecules. by analytical ultracentrifugation (20.8 kDa) and light-scattering Notably, EF-P is an acidic protein (calculated pI ϭ 4.6), and most experiments (23.2 kDa) (Fig. 2). This finding suggests that the of its surface is negatively charged (Fig. 3 A and B). Therefore, monomer is the major functional unit of EF-P, although we its overall shape is reminiscent of that of tRNA, although it is cannot exclude the possibility that EF-P dimerizes during some currently unclear which arm of EF-P corresponds to the acceptor BIOCHEMISTRY functional stage. or anticodon arm of tRNA (Fig. 3 A and B). The overall shape of the EF-P monomer (Figs. 1C and 3 A and The structures of the two EF-P monomers in the asymmetric B) is remarkably similar to the L shape of the tRNA molecule unit are quite similar to each other, with an rms deviation (rmsd) (Fig. 3C). One arm of the L, made by domains I and II (Fig. 1C), of 0.79 Å over all the protein atoms. Nevertheless, their inter- is Ϸ65 Å long and 23 Å wide (Fig. 3 A and B), whereas the other domain orientations differ slightly. Although the domain I arm, formed by domains II and III (Fig. 1C), is Ϸ53 Å long and structures of molecules A and B are practically the same (they 25 Å wide (Fig. 3 A and B). The angle made by the two arms of can be superposed on each other with an rmsd of 0.57 Å), the EF-P is Ϸ95° (Fig. 3 A and B). In the yeast tRNAPhe L-shaped relative orientation of domain I to domain II in the two structure, with two arms at Ϸ90° (28–30), the acceptor-T arm is molecules differs by Ϸ4°. Therefore, this arm is slightly flexible. Ϸ65 Å long and 22 Å wide, whereas the anticodon-D arm is Ϸ70 On the other hand, the difference in the relative orientation of Å long and 20 Å wide (Fig. 3C). The overall shapes and the sizes domains II and III between molecules A and B is negligibly small. of tRNA molecules are well conserved. The shape and the size Both of the domain I–II and II–III interfaces are formed by hydrophobic side chains with high surface complementarities. Therefore, the L shape of EF-P is likely to be a native confor- mation, rather than an artifact due to crystal packing.

Fig. 2. A plot of the sedimentation equilibrium data with the residuals from the best fit to a single ideal species. This plot shows the data with protein at Fig. 3. Structure comparison of EF-P with tRNA and ribosome-binding 0.5 mg⅐mlϪ1 and a speed of 20,000 rpm. The estimated partial specific volume proteins. (A and B) EF-P from T. thermophilus (PDB ID code 1UEB). (C) tRNAPhe of the protein is 0.74, and the solvent density was calculated to be 1.005 from Saccharomyces cerevisiae (PDB ID code 1EVV). (D) EF-G from T. ther- g⅐mlϪ1. All nine data sets (three speeds, three concentrations) were fitted mophilus (PDB ID code 1EFG). (E) Ribosome recycling factor from E. coli (PDB together. code 1EK8). (F) Release factor 2 from E. coli (PDB ID code 1GQE).

Hanawa-Suetsugu et al. PNAS ͉ June 29, 2004 ͉ vol. 101 ͉ no. 26 ͉ 9597 Downloaded by guest on October 1, 2021 Fig. 4. Alignment of the amino acid sequences of EF-P and eIF-5A. bTth, bEco, and bBsu are the bacterial EF-P proteins from T. thermophilus, E. coli, and Bacillus subtilis, respectively. eHum, eSce, ePae, and eMja are the eIF-5A proteins from Homo sapiens, S. cerevisiae, P. aerophilum, and M. jannaschii, respectively. The secondary structure of EF-P from T. thermophilus and M. jannaschii are indicated with arrows for ␤-strands and coils for helices. The amino acid residues conserved throughout the EF-P proteins are highlighted in yellow. The amino acid residues completely conserved throughout EF-P and eIF-5A are shown with white letters highlighted in red, and those well conserved in EF-P and eIF-5A are shown with red letters.

Several proteins possess domain(s) similar to a portion of side, but its other side is curved, and, together with the smaller tRNA. The C-terminal domain of EF-G, protruding from the ␤-sheet, it is involved in a ␤-barrel with a hydrophobic core. The ␤ ␤ globular GTPase domain, reportedly has a shape similar to that 310-helix is located between 1 and 2, forming the lid of the of the anticodon-stem loop in the EF-Tu–tRNA–GDPNP ter- barrel. The ␤3-, ␤4-, and ␤5-strands are much longer than nary complex (31, 32). Ribosome recycling factor, eukaryal the other strands, and therefore, the region connecting ␤3 and release factor 1, and release factor 2 each possess a protruding ␤4 (amino acid residues 28–36) protrudes outward. The con- domain (33–37). The entire structure of EF-P mimics the overall served basic residues, Arg-8, Lys-29, Arg-32, Lys-40, and Lys-42, shape of a tRNA molecule, like these tRNA-mimicking proteins are clustered on the ␤3-side surface of domain I (Fig. 3A), (Fig. 3). implying the significance of this region in EF-P function, such as However, the ‘‘tRNA mimicry’’ does not necessarily mean that nucleic acid binding. On the other hand, the opposite surface of the proteins bind to the tRNA-binding sites on the ribosome as domain I (Fig. 3B) is more negatively charged, as Asp-6, Asp-16, tRNA molecules do (36). Based on a cryo-electron microscopy and Glu-61 are highly conserved among the EF-P sequences analysis, it is reported that release factor 2 is incorporated in the (Fig. 4A). ribosome by assuming a different shape from that observed in It is remarkable that central domain II and C-terminal domain the crystal structure and by changing its interdomain orienta- III of EF-P possess the same fold (Fig. 1 B and C). The two tions (38, 39). A biochemical analysis revealed that the binding ␤-barrel domains are tandemly arranged along the axis of one mode of ribosome-recycling factor is different from that of arm of the L-shaped EF-P molecule. Domain II (amino acid tRNA (40). It has been hypothesized that the tRNA mimicry residues 65–126) contains five ␤-strands (␤7–␤11). The ␤7-, ␤8-, might allow the proteins to pass through the entrance of the ␤ ␤ ribosome (36). In this context, biochemical data suggested that and 9-strands form a curved antiparallel -sheet on one hand, whereas ␤7, ␤10, and ␤11 form a similar antiparallel ␤-sheet on EF-P binds to the A site of the ribosome (6, 7). Ganoza et al. (6) ␤ proposed that the EF-P-binding domain is very near the site of the other, and thus together they construct a typical -barrel EF-Tu and EF-G binding on both the 30S and 50S subunits. structure with a hydrophobic core. Domain III (amino acid ␤ However, the ribosome-binding manner of EF-P is unknown and residues 127–184) also possesses a -barrel architecture consist- ␤ ␤ ␤ must be studied further with the ribosome-bound state structure ing of five -strands ( 12– 16), in which two antiparallel ␤ ␤ ␤ ␤ ␤ ␤ ␤ of the EF-P. -sheets, consisting of 12, 13, and 14, and 12, 15, and 16, respectively, face each other. Thus, the two domains possess the Domain Architectures. The N-terminal domain I (amino acid same topology for the strand connectivities. Domains II and III residues 1–64) contains six ␤-strands (␤1–␤6) and a one-turn were superposed on each other with an rmsd of 1.2 Å for 31 C␣ ␤ ␤ ␤ ␤ 310-helix (Fig. 1 B and C). The strands 2, 3, 4, and 5 form atoms. These domains share partial sequence similarity (10 of 58 a large antiparallel ␤-sheet, whereas ␤1 and ␤6 form a smaller, residues are identical), which implies that they originated from curved antiparallel ␤-sheet. The larger ␤-sheet is flat on one a single domain, probably by a duplication event. It should be

9598 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0308667101 Hanawa-Suetsugu et al. Downloaded by guest on October 1, 2021 atoms). Consistent with the structural information, the domain I amino acid sequences of the known EF-P and eIF-5A proteins share some similarity (Fig. 4). According to a structure-based sequence comparison, 42% of the T. thermophilus EF-P amino acid residues are conserved or semiconserved in eIF-5As. In particular, the amino acid residues corresponding to Lys-29, Gly-31, Gly-33, and Ala-35 of the T. thermophilus EF-P are absolutely conserved in the EF-P͞eIF-5A superfamily, and they are located on the loop connecting ␤3 and ␤4 in both the EF-P and eIF-5A structures. The eIF-5As conserve a Lys residue at the tip of the loop (Lys-40, Lys-42, and Lys-37 for the M. jannaschii, P. aerophilum, and P. horikoshii eIF-5A proteins, respectively). This Lys residue is modified posttranslationally to a hypusine to generate the mature eIF-5A (12). It is remarkable that a Lys or Arg residue is also strictly conserved at the corresponding position of the EF-P proteins in bacteria (Fig. 4). For T. thermophilus EF-P, the corresponding basic residue is Arg-32. In the present EF-P crystal structure, the Arg-32 side chain pro- trudes toward the solvent at the end of the domain I arm. The bacterial EF-P reportedly lacks hypusine (13). Nevertheless, the conservation of the amino acid residues in the ␤3–␤4 connective linker and the basic residue at the tip of the loop implies the significance of this region for EF-P function. The eIF-5A C domain was compared with the EF-P domain II (Fig. 5A). The C domains of the M. jannaschii, P. aerophilum, Fig. 5. Structure comparison of EF-P and eIF-5A. (A) Superimposition of the and P. horikoshii eIF-5As overlapped well on the EF-P domain ribbon diagrams of T. thermophilus EF-P (blue) and M. jannaschii eIF-5A II, with rmsd values of 1.4 Å, 1.5 Å, and 2.0 Å, for 53, 58, and BIOCHEMISTRY (yellow). (B) Amino acid residues conserved in EF-Ps and eIF-5As color-coded 56 C␣ atoms, respectively. The similarity between the EF-P and on the surface of T. thermophilus EF-P. eIF-5A sequences exists only in the N-terminal half of domain II (the ␤7–␤9 region in EF-P) and not in the C-terminal half of domain II (Fig. 3). On the other hand, as the EF-P domains II noted here that the sequence of domain III is much better and III share the same folding topology, the eIF-5A domain II conserved than that of domain II in the EF-P proteins (Fig. 4). also superposed well on the EF-P domain III (rmsd values of 2.1 According to a DALI-based protein–structure comparison, Å, 2.4 Å, and 2.2 Å, for 41, 41, and 43 C␣ atoms of the eIF-5A the fold composed of domains II and III is similar to that of the proteins from M. jannaschii, P. aerophilum, and P. horikoshii, so-called oligonucleotide-binding fold, observed in E. coli cold- respectively). It is remarkable that the amino acid sequence of shock protein, the RNA-binding domains of E. coli polyribonu- the C-terminal half of the eIF-5A C domain is similar to that of cleotide nucleotidyltransferase, E. coli transcription factor Rho, the C-terminal half of EF-P domain III (the ␤15–␤16 region in Pyrococcus kodakaraensis aspartyl-tRNA synthetases, and so EF-P) (Fig. 5B). Thus, a gap appears in the eIF-5A sequences, ͞ forth. Thus, it is possible that domains II and or III of EF-P are as compared with the EF-P sequences. This implies the possi- involved in RNA (or DNA) binding. However, because almost bility that eIF-5A originated from an ancestral three-domain the entire surface of domain II is negatively charged (Glu-76, protein common to EF-P by a deletion event, in which the Glu-78, Asp-84, Glu-89, and Glu-106 are conserved), this do- ancestral eIF-5A might have lost the region corresponding to the main probably does not bind nucleic acids. In contrast, one EF-P ␤10–␤14 region. Because the missing region topologically surface of domain III has a patch of conserved basic residues, corresponds to a single ␤-barrel domain, the resultant eIF-5A C including Arg-140, Lys-149, Arg-176, and Arg-183, which are domain retains the same folding topology as domains II and III probably favorable for nucleic acid binding. The other surface of of EF-P. On the other hand, it is also possible that the sequence domain III is negatively charged, and Asp-134, Glu-154, Glu-166, of the EF-P ␤10–␤14 region diversified after domains II and III and Glu-169 are conserved. were formed by duplication.

Comparison Between EF-P and eIF-5A. eIF-5A is an archaeal͞ Concluding Remarks. The overall tRNA-like shape of the EF-P eukaryal paralog of EF-P. Thus far, three eIF-5A crystal struc- molecule and its charge distribution seem to be suitable for this tures have been reported, from M. jannaschii, P. aerophilum, and protein to bind to the ribosome by spanning the two subunits (6). P. horikoshii (14–16). These structures revealed that eIF-5A EF-P may bind to the tRNA-binding site(s) on the ribosome by consists of only two ␤-barrel domains. These N and C domains mimicking the tRNA shape. eIF-5A corresponds to domains I appear to correspond to domains I and II (or III), respectively, and II of EF-P. It is interesting to note that, in this context, of EF-P. The overall shape of the two-domain eIF-5A is a eIF-5A might correspond to a minihelix or anticodon helix of straight bar, in contrast to the L-shaped structure of the three- tRNA. Although eIF5A is not thought to be essentially involved domain EF-P (Fig. 5A). Intriguingly, slight flexibility in the in translation in yeast (11), it is also possible that, in Archaea and relative orientation of domain I to domain II has also been found Eukarya, some other protein or RNA factor(s) compensates for the M. jannaschii and P. horikoshii eIF-5A proteins (Ϸ7°) (14, structurally or functionally for the missing third domain. Many 16), which is very similar to the internal-domain flexibility of questions about EF-P still remain. How does EF-P interact with EF-P (Ϸ4°) described above. the ribosome? Which arm of the L corresponds to the acceptor The structure of the EF-P domain I superposed well on those or anticodon arm of tRNA? Does it form a ternary complex with of the N domains of M. jannaschii eIF-5A (rmsd ϭ 1.3 Å per 61 EF-Tu⅐GTP? How can it activate the peptidyltransferase of the C␣ atoms), P. aerophilum eIF-5A (rmsd ϭ 1.2 Å per 60 C␣ ribosome? To answer these questions, further functional and atoms), and P. horikoshii eIF-5A (rmsd ϭ 1.4 Å per 63 C␣ structural studies are needed.

Hanawa-Suetsugu et al. PNAS ͉ June 29, 2004 ͉ vol. 101 ͉ no. 26 ͉ 9599 Downloaded by guest on October 1, 2021 We thank R. Ushikoshi, H. Tanaka, and Y. Kamewari for preparation work was supported in part by a grant from the Organized Research of the T. thermophilus EF-P protein, H. Nakajima and Drs. Y. Kawano Combination System of the Science and Technology Agency of Japan and N. Kamiya for supporting our data collection at beamline 45PX at and by the RIKEN Structural Genomics͞Proteomics Initiative and the SPring-8, and Dr. S. Yokobori (Tokyo University of Pharmacy and Life National Project on Protein Structural and Functional Analyses, Min- Science) for helpful discussions about the phylogenetic analysis. This istry of Education, Culture, Sports, Science and Technology of Japan.

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