Crystal structure of YlqF, a circularly permuted GTPase: Implications for its GTPase activation in 50 S ribosomal subunit assembly



Members of the GTPase superfamily play important roles in diverse cellular processes such as cell division, cell cycling, signal transduction, mRNA translation (initiation, elongation, and termination), and ribosome assembly.1 They have been divided into two large classes, TRAFAC (named after translation factors) and SIMBI (after signal recognition particle, MinD, and BioD).2 One of the distinct families of TRAFAC class GTPases is the YlqF/YawG family, which consists of five distinct subfamilies, typified by the proteins YlqF (from Bacillus subtilis), YqeH (B. subtilis), YjeQ (Escherichia coli), MJ1464 (Methanocaldococcus jannaschii), and YawG (Schizosaccharomyces pombe). A special feature of this YlqF/YawG family is a circular permutation of the GTPase signature motifs.2 Members of the YlqF subfamily are broadly conserved in eukaryotes, archaea, and bacteria.3 Several members of this subfamily from human (NGP-1 and nucleostemin) and yeast (Nug1p, Nug2p, and Nog2p) have been shown to localize to the nucleolus, and some of them are closely associated with ribosomal assembly and nucleolar/nuclear export of ribosomal subunits.4 One member of this group, nucleostemin, was preferentially expressed in nucleoli of central nervous system stem cells, embryonic stem cells, and other cancer cell lines, and may control cell-cycle progression.4

Recently, multiple GTP-binding proteins have been implicated in the assembly of bacterial ribosomes. Of the 13 essential GTP-binding proteins in B. subtilis, seven (Era, Obg, YphC, YsxC, YlqF, YqeH, and YloQ) have been reported to associate with the 50 S or 30 S ribosomal subunits.5 In B. subtilis, the circularly permuted GTPase YlqF participates in the late step of 50 S subunit assembly and is essential for cell viability.6 It was proposed to rename the ylqF gene rbgA (ribosome biogenesis GTPase A).6B. subtilis YlqF was shown to be targeted to the premature 50 S subunit lacking ribosomal proteins L16 and L27 to assemble a functional 50 S subunit through a GTPase activity-dependent conformational change of 23 S rRNA.7 The GTPase activity of B. subtilis YlqF is stimulated by binding of the premature 50 S subunit or the 50 S subunit.6, 7B. subtilis YlqF can bind stably to the free 50 S subunit in the presence of the nonhydrolyzable GTP analog GTPγS, suggesting that a possible conformational change from the GTP- to GDP-bound form is important for the dissociation of YlqF from the mature 50 S subunit.7 Despite these recent findings, little is known about the activation mechanism of the YlqF GTPase activity and the nature of the possible conformational change that accompanies GTP hydrolysis. Therefore, structure determination of YlqF bound with GTP and GDP is essential for the elucidation of the activation mechanism of the YlqF GTPase activity and for understanding how YlqF assists in the biogenesis of the 50 S ribosomal subunit.

To provide the structural basis for a better understanding of the biochemical functions of YlqF GTPase subfamily members, we have determined the crystal structure of YlqF from Thermotoga maritima (Tm) in complexes with GDP, GTP, and a nonhydrolyzable GTP analog, guanosine 5′-[β, γ-imido] triphosphate (GppNHp; abbreviated here as GNP). Tm YlqF shows 31% sequence identity to B. subtilis YlqF and 23% to the GTPase domain of human nucleostemin. The structure of Tm YlqF is composed of two domains: an N-terminal GTPase domain and a C-terminal helical domain. The structures of Tm YlqF bound with different ligands reveal a significant difference in the relative orientation of the two domains, when we compare the GDP-bound structure with either the GTP- or GNP-bound structure. Our study thus provides a glimpse of a possible conformational change of YlqF upon GTP hydrolysis. It also sheds light on the activation mechanism of the YlqF GTPase activity in the 50 S ribosomal subunit assembly.


Protein expression and purification

The ylqF gene from T. maritima (Tm_0768) was cloned into the expression vector pET-21a(+) (Novagen). The recombinant protein without any fusion tag was overexpressed in E. coli Rosetta2(DE3)pLysS cells using terrific broth culture medium. Protein expression was induced by 0.5 mM isopropyl 1-thio-β-D-galactopyranoside, and the cells were incubated for an additional 6 h at 37°C following growth to mid-log phase at 37°C. The cells were lysed by sonication in buffer A (20 mM Tris–HCl at pH 7.5) with 200 mM NaCl and 10% (v/v) glycerol. After heat treatment at 80°C for 10 min, the sample was centrifuged at 3,800g for 60 min. The supernatant was diluted twofold with buffer A and applied to a HiLoad 26/10 Q-Sepharose column (GE Healthcare), which was previously equilibrated with buffer A. Upon eluting with a gradient of NaCl in the same buffer, YlqF was eluted at 250–300 mM NaCl concentration. The protein was further purified by gel filtration on a HiLoad XK-16 Superdex 200 prep-grade column (GE Healthcare), which was previously equilibrated with buffer A with 200 mM NaCl. Fractions containing YlqF were concentrated to 6 mg mL−1 for crystallization using an Amicon Ultra-15 centrifugal filter unit (Millipore).

Crystallization and X-ray data collection

Crystals were grown by the hanging-drop vapor diffusion method at 24°C by mixing equal volumes (2 μL each) of the protein solution (at 6 mg mL−1 concentration in buffer A with 200 mM NaCl) and the reservoir solution. Crystals were grown with a reservoir solution consisting of 100 mM HEPES at pH 7.5, 5% (v/v) isopropanol, and 20% (w/v) polyethylene glycol 4000. Rod-shaped crystals grew to approximate dimensions of 0.1 mm × 0.1 mm × 0.3 mm within a few days. Ligand-bound crystals were obtained by cocrystallization in the presence of 1.5 mM Mg2+ and 1.5 mM each of GNP, GTP, or GDP.

A crystal was dipped into a cryoprotectant solution for a few seconds and frozen in the cold nitrogen gas stream at 100 K. The cryoprotectant solution consisted of 20% (v/v) glycerol added to the reservoir solution. X-ray diffraction data were collected at 100 K on a Quantum 4R CCD detector (Area Detector Systems Corporation, Poway, CA) at the BL-6C experimental station of Pohang Light Source, Korea. The raw data were processed and scaled using the program suit HKL2000.8 The GNP-cocrystallized crystal belongs to the space group P212121, with unit cell parameters of a = 35.13 Å, b = 75.15 Å, and c = 104.92 Å. There is one monomeric YlqF molecule per asymmetric unit, giving a solvent fraction of 44.6%. Other X-ray diffraction data were collected essentially as mentioned earlier. Table I summarizes data collection statistics.

Table I. Statistics for Crystallographic Data Collection and Structure Refinement
X-ray wavelength (Å)1.23985 (Pohang light source beamline 6C)
  • a

    Values in parentheses refer to the highest resolution shell.

  • b

    Rmerge = Σh Σi | I(h)i – 〈I(h)〉 | /Σh ΣiI(h)i, where I(h) is the intensity of reflection h, Σh is the sum over all reflections, and Σi is the sum over i measurements of reflection h.

  • c

    R = Σ | |Fo| – |Fc| | /Σ |Fo|, where Rfree is calculated for a randomly chosen 10% of reflections, which were not used for structure refinement, and Rwork is calculated for the remaining reflections.

Data collection
 Data setGNP complexGTP complexGDP complex
 Space groupP212121P212121P212121
 a (Å)35.1335.2634.22
 b (Å)75.1575.3076.66
 c (Å)104.92105.11104.90
 Resolution range (Å)20–2.0 (2.07–2.0)a20–2.3 (2.38–2.3)a20–2.3 (2.38–2.3)a
 Total/unique reflections129,724/19,46081,457/12,90389,197/12,683
 Completeness (%)99.6 (96.7)a98.7 (89.4)a99.2 (92.6)a
 II32.2 (3.2)a32.3 (2.7)a25.6 (2.7)a
 Rmergeb0.058 (0.417)a0.064 (0.340)a0.077 (0.412)a
Model refinement
 Number of atoms/average B-factor (Å2)
  Protein nonhydrogen atoms1,870/35.91,824/50.01,824/44.0
  Water oxygen atoms143/42.391/56.6114/48.2
  Ligand atoms32/29.232/44.628/29.5
 R.m.s. deviations from ideal geometry
  Bond lengths (Å)/bond angles (°)0.011/1.450.011/1.370.009/1.37
 Ramachandran plot (non-Gly and non-Pro)
  Most favorable (%)92.788.695.0
  Allowed (%)5.810.95.0
  Generously allowed (%)1.00.50
  Disallowed (%)0.500

Structure determination

The structure of the GNP complex was determined by molecular replacement, using a monomoer model of the GNP-bound structure of YlqF from B. subtilis (PDB code 1PUJ; unpublished deposition by the New York Structural Genomics Research Consortium) as a search model. Cross-rotational search, followed by translational search, was performed using the program PHASER.9 Subsequent manual model building was done using the program COOT.10 The model was refined with the program REFMAC,11 including the bulk solvent correction. Ten percentage of the data were randomly set aside as the test data for the calculation of Rfree.12 Subsequently, this refined model was used to refine the GTP-bound form and the GDP-bound form. All the models have excellent stereochemistry, as evaluated by the program PROCHECK.13 When we did not add any guanine nucleotide during crystallization, GDP was bound in the active site. This set of data is not included in Table I.

Data deposition

The atomic coordinates and structure factors of Tm YlqF have been deposited in the Protein Data Bank under accession codes 3CNL (GNP complex), 3CNN (GTP complex), and 3CNO (GDP complex).


Overall structure and structural comparisons

The structure of the GNP complex of Tm YlqF was refined against 20–2.0 Å data to Rwork and Rfree values of 0.231 and 0.280, respectively. The refined model accounts for 233 residues (residues 9–124 and 135−251) of the 262 residues in Tm YlqF. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the polypeptide chains recovered from the crystal are not cleaved into smaller fragments, indicating that the missing residues in the middle (residues 125−134) are disordered in the crystal. We have additionally determined the structures of Tm YlqF bound with either GTP or GDP. The structure of the GTP complex was refined against 20–2.3 Å data to Rwork and Rfree values of 0.229 and 0.283, respectively. The structure of the GDP complex was refined against 20–2.3 Å data to Rwork and Rfree values of 0.215 and 0.276, respectively. The refined models of both GTP and GDP complexes account for 227 amino acid residues (13–124 and 136−250). Refinement statistics are summarized in Table I. Lys12 is the only outlier in the Ramachandran plot of the GNP complex (Table I). It is poorly defined by the electron density. The residues Glu9–Lys12 of the GNP complex are disordered in the GTP and GDP complexes.

Tm YlqF exists as a monomer in solution according to our dynamic light scattering measurements. Tm YlqF has approximate dimensions of 64 Å × 32 Å × 26 Å [Fig. 1(A)] and consists of two domains: an N-terminal GTPase domain (residues 1−156) and a C-terminal helical domain (residues 157−262). The N-terminal GTPase domain of Tm YlqF has the characteristic Rossman fold, resembling other members of the TRAFAC GTPase superfamily.2 The C-terminal domain contains four α-helices (α5–α8) and a 310-helix that interact hydrophobically with each other, forming a globular α-helical bundle, and a protrusion between α7 and α8 forms a β-hairpin [Figs. 1(A,B)].

Figure 1.

Overall fold of Tm YlqF and electron density of the bound ligands. A: Ribbon diagram of Tm YlqF. α-Helices, β-strands, and loops are colored in salmon, blue, and white, respectively. The bound GNP is shown in a stick model. All the figures except Figs. 1(B) and 2 are drawn with PyMOL (DeLano, 2002, The PyMOL Molecular Graphics System, B: Topology diagram of Tm YlqF. β-Strands are shown as arrows and α-helices as cylinders. C:Fo − Fc omit electron density maps of the bound ligands. D: Stereo Cα trace of Tm YlqF. Every tenth residue is marked by a black dot and every twentieth residue is labeled. Conserved sequence motifs are highlighted in colored lines: motif G5* [DARhP, residues 30–34 of Tm YlqF] in orange, motif G4 [LNKXD, residues 53–57] in green, motif G1 [GhPNXGKST, residues 106–114] in red, motif G3 [DTPG, residues 150–153] in blue, the visible part (residues 9−13) of the conserved N-terminal sequence motif [W(F/Y)PGH(M/I)XKA(K/R)R] in cyan, and the conserved sequence motif of the C-terminal domain [KRG] at positions 212–214 in pink, respectively. Motif G2* [GhT, residues 132–134] is disordered in the crystal.

The closest homologs of Tm YlqF N-terminal domain (GNP-complex), as identified by a DALI structural similarity search,14 were B. subtilis YlqF (PDB code 1PUJ) (a Z-score of 16.0, a root-mean-square (r.m.s.) deviation of 2.0 Å for 133 equivalent Cα positions, and a sequence identity of 32%) and Tm YjeQ (PDB code 1U0L)15 (a Z-score of 13.4, an r.m.s. deviation of 2.7 Å for 129 equivalent Cα positions, and a sequence identity of 19%). A DALI search with the C-terminal domain of Tm YlqF (GNP-complex) identified B. subtilis YlqF (PDB code 1PUJ) (a Z-score of 5.9, an r.m.s. deviation of 2.2 Å for 87 equivalent Cα positions, and a sequence identity of 22%) as the only close homolog. The next highest Z-score is only 3.0 with a putative antiterminator from Mycobacterium tuberculosis (PDB code 1S8N; an r.m.s. deviation of 3.1 Å for 52 equivalent Cα positions and a sequence identity of 13%).16 YjeQ is another circularly permuted GTPase subfamily of the TRAFAC superfamily and is known to bind to the ribosome.15 Unlike YlqF, Tm YjeQ is composed of three distinct domains: an N-terminal OB-fold domain, a central circularly permutated GTPase domain, and a C-terminal zinc-finger domain.

Guanine nucleotide binding and circularly permuted G motifs

The GNP, GTP, and GDP molecules are bound to the N-terminal GTPase domain near the cleft between the N-terminal and C-terminal domains (Fig. 1). However, the C-terminal domain does not interact with the ligands. The nonhydrolyzed GTP bound to the active site of Tm YlqF confirms the low intrinsic GTPase activity of Tm YlqF, which is consistent with the activation of the B. subtilis YlqF GTPase activity by binding of the premature 50 S subunit.5

Five sequence motifs contribute directly or indirectly to the formation of the guanine nucleotide-binding pocket on the surface of the N-terminal GTPase domain [Fig. 1(D)]. In the circularly permuted Tm YlqF, these sequence motifs occur in the following order: G5*-G4-G1-G2*-G3 [Figs. 1(B) and 2].2, 17 The G5 and G2 sequence motifs are not well conserved among GTP-binding proteins, and the amino acid residues DARhP and GhT have been referred to as G5* and G2*, respectively,4, 18 where “h” stands for a hydrophobic residue.

Figure 2.

Sequence alignment of bacterial YlqF proteins. Tm, T. maritima (SWISS-PROT accession code: Q9WZM6); Bs, B. subtilis (O31743); Bh, Bacillus halodurans (Q9Z9S1); Sa, Staphylococcus aureus (Q7A5Y9). Cylinders above the sequences denote α-helices and arrows β-strands. Conserved sequence motifs are enclosed by colored boxes: motif G5* [DARhP, residues 30–34] in orange, motif G4 [LNKXD, residues 53–57] in green, motif G1 [GhPNXGKST, residues 106–114] in red, motif G2* [GhT, residues 132–134] in purple, motif G3 [DTPG, residues 150–153] in blue, the N-terminal conserved sequence motif [W(F/Y)PGH(M/I)XKA(K/R)R, residues 3–13] in cyan, and the conserved sequence motif of the C-terminal domain [KRG, residues 212–214] in pink, respectively. This figure was drawn with ClustalX19 and GeneDoc (

The sequence motif G5* [DARhP] encompasses Asp30–Pro34 of Tm YlqF [boxed in orange in Fig. 2; highlighted in orange lines in Fig. 1(D)]. The conserved motif G5* does not directly interact with the bound GNP, GTP, or GDP. Instead, the OD2 atom of Asp30 interacts with the side chains of Lys55 from the G4 motif and Asn109 from the G1 motif via a water molecule, contributing to the stability of the guanine nucleotide-binding pocket. The OD1 and OD2 atoms of Asp30 form hydrogen bonds with the NE and NH2 nitrogen atoms of the conserved Arg32 in the G5* motif, enhancing the rigidity of the conserved G5* loop region.

The G4 motif [LNKXD] covers Leu53–Asp57 of Tm YlqF [boxed in green in Fig. 2; highlighted in green lines in Fig. 1(D)] and is located near the guanine base of the bound ligand. “X” stands for any amino acid. The side chain of conserved Asp57 from G4 forms hydrogen bonds with the nitrogen atoms of the bound guanine base [Fig. 3(A)], controlling the specificity for guanine nucleotides like other canonical GTPases.20 The side chains of Asn54 and Lys55 make hydrogen bonds with the main-chain carbonyl oxygen atoms of Thr110 and Asn109 of the G1 motif, respectively.

Figure 3.

Binding of guanine nucleotides to Tm YlqF and domain movement. A: Interactions between protein atoms and the bound ligands. Black dotted lines denote hydrogen bonds. GNP and GDP are shown in sticks, and the residues belong to motif G2* and G4 are boxed in green and red, respectively. B: Stereo view of the active site around the β- and γ-phosphates of the bound GNP in Tm YlqF. A water molecule is represented as a red ball. C: Electrostatic potential at the surface of Tm YlqF. Blue and red colors correspond to positive and negative potentials, respectively. The bound GNP is shown in a stick model, and a black arrow indicates the visible part of the N-terminal conserved sequence motif (boxed in cyan in Fig. 2). D: Stereo view of the superimposed structures of GNP- and GDP-bound Tm YlqF (colored in green and yellow, respectively). This illustrates the ligand-dependent domain movement.

The conserved motif G1 [GhPNXGKST] [boxed in red in Fig. 2; highlighted in red lines in Fig. 1(D)], which is also referred to as Walker A motif or the P-loop, covers Gly106–Thr114 of Tm YlqF. This motif encircles the charged phosphate groups of the bound ligand [Fig. 3(A)], and the side chain of the conserved lysine residue (Lys112) of the P-loop directly interacts with the oxygen atoms of the β- and γ-phosphates of GNP and GTP [Fig. 3(A)]. In the GDP-bound structure, a water molecule replaces the γ-phosphate of GTP or GNP and forms a hydrogen bond with the NZ nitrogen atom of Lys112, as well as with the O3B oxygen atom of the β-phosphate [Fig. 3(A)]. Together with the side-chain of Lys112, the main-chain nitrogen atoms of Asn109, Gly111, Lys112, and Thr114 make hydrogen bonds with the phosphate oxygen atoms of the bound ligand [Fig. 3(A)].

The G2* motif [GhT], covering the switch-I region, corresponds to Gly132–Thr134 of Tm YlqF [boxed in purple in Fig. 2]. It is disordered in all three structures of Tm YlqF [Figs. 1(A,D)]. It is also disordered in the structures of other circularly permuted GTPases: GDP-bound Tm YjeQ15 and GNP-bound YlqF from B. subtillis (PDB code 1PUJ). The flexibility of this motif may be required for a conformational change of YlqF upon GTP hydrolysis. Thr35 of the corresponding G2 motif of the human ras protein interacts with the γ-phosphate in the GTP analog complexes and triggers a conformational change upon GTP hydrolysis.20 The loop Gly121–Gln138 containing the G2* motif of Tm YlqF is considerably elongated, compared to the structurally equivalent loop (His27–Glu37) of the human ras protein, possibly contributing to the flexibility of this loop region.

The G3 motif [DTPG], covering the switch-II region or the Walker B motif, encompasses Asp150–Gly153 of Tm YlqF [boxed in blue in Fig. 2; highlighted in blue lines in Fig. 1(D)]. This motif does not interact directly with the bound ligand in any of the Tm YlqF structures. The side chain of the conserved Asp150 points away from the active site in all structures of Tm YlqF, irrespective of the presence of the γ-phosphate in the ligand, due to the interaction between Asp150 and Arg72 from a symmetry-related molecule in the crystal [Fig. 3(B)]. In comparison, the corresponding residue (Asp171) in the B. subtilis YlqF switch-II region binds Mg2+ through a water molecule (PDB code 1PUJ).

The GTPase activity of YlqF is stimulated by binding of the premature 50 S ribosomal subunit.7 The concave surface of the guanine nucleotide binding side of Tm YlqF is highly electropositive [Fig. 3(C)], and it may be important in binding 23 S rRNA. If this is the case, the switch-I and II regions could become ordered upon binding of the premature 50 S subunit, and the ordering of these switch regions may be a prerequisite for the activation of the GTPase activity. A similar ordering was demonstrated in the cryo-EM reconstruction of the 70 S·EF-G ribosomal complex, in which ordering of the functionally important switch-I and II regions of EF-G is dependent on interactions with the ribosome.21

Other sequence motifs of YlqF

Besides the G1–G5 motifs, a highly conserved sequence motif [W(F/Y)PGH(M/I)XKA(K/R)R] [boxed in cyan in Fig. 2; highlighted in cyan lines in Fig. 1(D); indicated by an arrow in Fig. 3(C)], corresponding to Trp3–Arg13 of Tm YlqF, is present at the N-terminus of YlqF subfamily members. In the GNP-bound structure of Tm YlqF, part of this motif (residues 9–13) is visible in the electron density map and form a loop and the start of the first helix α1. Recently, it was reported that the GTPase activity of the N-terminal deletion mutant of YlqF (YlqFΔN10) that lacks the first 10 residues could not be activated by the 50 S subunit so that the mutant protein could not dissociate from the premature 50 S subunit.7 This suggests that one or more of the conserved residues in the first 10 residues of B. subtilis YlqF play an essential role in the activation of its GTPase activity by providing one or more of the key catalytic residues into the active site.

In the active site of several GTPases, a key glutamine residue is in close proximity to the γ-phosphate, playing a catalytic role.22 In the case of Arf GTPase, the Arf GTPase activating protein provides the catalytic histidine.22 It has been noticed that B. subtilis YlqF has the catalytic glutamine or histidine substituted by a hydrophobic amino acid, and its switch-II region takes a “retracted” conformation unlike in classical GTPases.23 To activate the GTPase activity of YlqF, therefore, the key catalytic residue must be supplied by another protein or by the region other than the switch-II.23 One possible candidate is His9 of B. subtilis YlqF (corresponding to His7 of Tm YlqF), which is located close to the γ-phosphate (PDB code 1PUJ). A small conformational change involving the first helix α1 may occur upon binding of the premature 50 S subunit, and it may bring this histidine into close proximity to the γ-phosphate so that it could play the catalytic role. The sequence [XKA(K/R)R] that was not deleted in the B. subtilis N-terminal deletion mutant (YlqFΔN10) may provide the second key catalytic residue (likely an arginine) for the activation of the GTPase activity. Alternatively, it may play a role in binding 23 S rRNA, and the second key catalytic residue (likely an arginine) may be supplied by the premature 50 S subunit.

The C-terminal helical domain of YlqF is much less conserved than the N-terminal GTPase domain (see Fig. 2), with only a strictly conserved tripeptide sequence [KRG] corresponding to Lys212(Arg213–Gly214) of Tm YlqF [boxed in pink in Fig. 2; highlighted in pink lines in Fig. 1(D)]. This conserved sequence motif is located on the guanine nucleotide-binding side of Tm YlqF between helix α7 and the following β-hairpin. The side chain of Arg213 makes hydrogen bonds with the side chain of Glu234 on helix α8 of the C-terminal domain. Glu234 is conserved as Glu or Asp among YlqF members. These interactions may stabilize the conformation of the C-terminal domain, which contributes to the extended positively charged surface on the guanine nucleotide-binding side of Tm YlqF [Fig. 3(C)] and may play an important role in binding the premature 50 S ribosomal subunit.

Ligand-dependent domain movement

GTP- and GNP-bound structures of Tm YlqF are virtually identical, with an r.m.s. deviation of 0.16 Å for 227 Cα atoms. However, the GDP-bound model shows significantly larger structural differences when it is compared with the GTP- or GNP-bound structures, with r.m.s. deviations of 0.78 Å or 0.79 Å for 227 Cα atoms, respectively. The largest deviation occurs at the tip of the protruding β-hairpin between strands β7 and β8, with a maximum deviation of 4.2 Å for the Cα atom of Gly219. This is due to a significant difference in the relative domain orientation of the GDP-bound YlqF compared with those of the GTP-bound and GNP-bound structures.

The ligand-dependent domain rearrangement is more obvious when we overlap the individual domains. The structures of individual domains are highly similar among the GDP-, GTP-, and GNP-bound structures. The r.m.s. deviations range between 0.13 Å (GTP-complex vs. GNP-complex) and 0.27–0.28 Å (GDP-complex vs. GNP- or GTP-complex) for the 123 Cα atoms in the N-terminal domain. The r.m.s. deviations range between 0.17 Å (for GTP-complex vs. GNP-complex) and 0.72–0.77 Å (for GDP-complex vs. GNP- or GTP-complex) for the 94 Cα atoms in the C-terminal domain. The C-terminal domain is slightly more open in the GDP-bound structure [Fig. 3(D)], resulting in a maximum deviation of 6.3–6.4 Å for the Cα atom of Gly219 relative to GTP- or GNP-complexes, when we superimpose the N-terminal domains only. In contrast, the deviation for the Cα atom of Gly219 between GNP- and GTP-complexes is only 0.4 Å, when we superimpose the N-terminal domains only. This kind of domain rearrangement could play an important role in the dissociation of the mature 50 S ribosomal subunit from YlqF after GTP hydrolysis. There is a possibility that the observed domain movement in the crystal may have been restricted by the crystal packing forces.


We thank the staff at beamline BL-6C of Pohang Light Source, Korea, for assistance during X-ray experiments. DJK and JYJ are recipients of the BK21 fellowship.