JBC Transcription and Nuclear Factor Monoclonals

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Originally published In Press as doi:10.1074/jbc.M607830200 on November 2, 2006

J. Biol. Chem., Vol. 282, Issue 1, 657-666, January 5, 2007
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Sordarin Derivatives Induce a Novel Conformation of the Yeast Ribosome Translocation Factor eEF2*

Rikke Søe{ddagger}, Ralph T. Mosley§, Michael Justice, Jennifer Nielsen-Kahn, Mythili Shastry, A. Rod Merrill||1, and Gregers R. Andersen{ddagger}2

From the {ddagger}Centre for Structural Biology, Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark, the Departments of §Medicinal Chemistry and Infectious Diseases, Merck Research Laboratories, Rahway, New Jersey 07065, and the ||Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada

Received for publication, August 16, 2006 , and in revised form, November 2, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The sordarins are fungal specific inhibitors of the translation factor eEF2, which catalyzes the translocation of tRNA and mRNA after peptide bond formation. We have determined the crystal structures of eEF2 in complex with two novel sordarin derivatives. In both structures, the three domains of eEF2 that form the ligand-binding pocket are oriented in a different manner relative to the rest of eEF2 compared with our previous structure of eEF2 in complex with the parent natural product sordarin. Yeast eEF2 is also shown to bind adenylic nucleotides, which can be displaced by sordarin, suggesting that ADP or ATP also bind to the three C-terminal domains of eEF2. Fusidic acid is a universal inhibitor of translation that targets EF-G or eEF2 and is widely used as an antibiotic against Gram-positive bacteria. Based on mutations conferring resistance to fusidic acid, cryo-EM reconstructions, and x-ray structures of eEF2, EF-G, and an EF-G homolog, we suggest that the conformation of EF-G stalled on the 70 S ribosome by fusidic acid is similar to that of eEF2 trapped on the 80 S ribosome by sordarin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The elongation phase of translation consists of three steps (reviewed in Ref. 1). First, elongation factor 1A (eEF1A in eukaryotes and EF-Tu in prokaryotes) delivers aminoacyl-tRNA to the ribosomal A-site. Second, the peptidyl transferase activity within the large subunit of the ribosome catalyzes the rapid transfer of the peptidyl moiety from the peptidyl-tRNA in the ribosomal P-site to the aminoacyl-tRNA at the A-site with the formation of the peptide bond. Finally, the deacylated tRNA in the P-site and the peptidyl-tRNA in the A-site must be translocated to the E- and P-site, respectively. The mRNA must also translocate by exactly three bases to maintain the reading frame and expose the next codon in the decoding site of the small subunit. The translocation reaction is catalyzed by elongation factor 2 (eEF2 in eukaryotes and EF-G in prokaryotes). In fungi, an additional elongation factor, eEF3, is required to facilitate release of the deacylated tRNA from the E-site (2). This may be considered as an extension to the normal translocation reaction, because it prepares the fungal 80 S ribosome for delivery of the next aminoacyl-tRNA to the A-site. Although both eEF1A and eEF2 use the energy derived from GTP hydrolysis for function, the activity of eEF3 depends on its hydrolysis of ATP (3).

Translocation involves the coordinated movement of three macromolecules relative to the ribosome and is therefore likely to be rather complex. The mechanism of translocation is currently not well defined, although thorough kinetic studies of how EF-G promotes translocation on 70 S ribosomes have been conducted (4-7). Advanced kinetic studies of the eukaryotic system are currently lacking, but the fundamental mechanism is believed to be conserved between the kingdoms, and both the ribosome and the elongation factor have high structural homology. This has been confirmed by cryo-EM3 reconstructions of eEF2 in a sordarin-stabilized complex with the 80 S ribosome (8, 9), which is rather similar to the 70 S-EF-G complex stabilized by fusidic acid (10). Both antibiotics prevent the release of the elongation factor from the ribosome after translocation (11). In both cases domain IV of eEF2/EF-G protrudes into the A-site of the small ribosomal subunit, and the location of the remaining domains is also quite similar for eEF2 and EF-G (9, 10). The function of EF-G/eEF2 appears to be dual, prior to translocation it promotes conformational changes within the pretranslocational ribosome-tRNA-mRNA complex, and after translocation it prevents peptidyl-tRNA from slipping back from the P-site. Conformational flexibility within EF-G is required for translation but apparently not for GTP hydrolysis (12). A detailed kinetic scheme has recently been derived for bacterial translocation (4). The main features of this are that GTP hydrolysis precedes a conformational change (unlocking) of the 70 S-EF-G complex required for translocation. In other studies, the state of the P-site tRNA is shown to govern the structural properties of the ribosome (5, 13). When deacylated tRNA, but not peptidyl-tRNA, is present conformational changes within the small subunit and its rotation relative to the large subunit are induced upon binding of EF-G (13). A similar rotation is observed when eEF2 binds to the 80 S ribosome (9). Potentially this rotation may contribute significantly to the physical movement of the two tRNA molecules, but several studies have also shown stringent requirements for the structural integrity of the two tRNA molecules to obtain efficient and in-frame translocation (reviewed in Ref. 14). A model for the movement of tRNA during translocation based on crystal structures, hydroxyl radical probing, and RNase protection experiments (15) suggests that peptide bond formation induces movement of the newly formed peptidyl-tRNA from the classical A/A state to a hybrid A/P state, where the anticodon stemloop is still located in the 40 S A-site, whereas the acceptor stem and end is in the 60 S P-site. In the same manner, the deacylated tRNA has moved to the P/E hybrid state. The existence of the P/E state on the 70 S ribosome has been confirmed by cryo-EM (13), whereas only subtle movements of the peptidyl-tRNA have been observed so far by either cryo-EM or crystallography.

Sordarin is a natural product originally isolated from the fungus Sordaria areneosa (16). The biochemical mechanism of action underlying the antifungal activity of sordarin has been elucidated (11, 17, 18). Sordarins are selective inhibitors of fungal protein synthesis and impair the function of eEF2. The binding of sordarin to eEF2 is greatly enhanced in the presence of ribosomes. Genetic studies in Saccharomyces cerevisiae have established that resistance to sordarin is conferred by mutations in eEF2 or the ribosomal stalk protein rpP0, although eEF2 is the principal determinant of sordarin specificity (11, 19-21).

The in vitro antifungal efficacy of sordarin and its analogs varies among species of pathogenic fungi (17, 22). For example, Candida albicans, Candida tropicalis, Candida glabrata, and Cryptococcus neoformans are quite sensitive to these compounds, whereas Cryptococcus parapsilosis and Cryptococcus krusei are relatively insensitive. Growth inhibition and in vitro translation assays using chimeric Candida-S. cerevisiae eEF2 proteins expressed in S. cerevisiae, have identified three amino acid residues in the protein (Tyr521, Ser523, and Glu524 in yeast eEF2) that are critical determinants of the spectrum of antifungal activity of sordarin (23). Modified sordarins, including synthetic analogs such as GM 237354 (22) and natural product analogs (24) have enhanced potency and antifungal spectrum of activity.

In this report, we describe the crystal structures of eEF2 in complex with two modified sordarins, both of which induce a conformation within eEF2 that is quite distinct from that described earlier for eEF2 in the presence of sordarin (25). Mammalian eEF2 has previously been shown to bind adenylic nucleotides in a site distinct from the well characterized binding site for GDP/GTP (26). We show that this is a universal feature of eEF2 and that sordarin can displace both ADP and ATP from eEF2. This suggests that either the binding site of sordarin overlaps with that of ADP/ATP or that sordarin induces a conformation in eEF2, which is incompatible with binding of ADP/ATP. Finally, we suggest that the conformation of EF-G on the prokaryotic ribosome targeted by fusidic acid is similar to that of the eEF2-sordarin complex on the eukaryotic ribosome.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Structure Determination and Modeling—Purification and crystallization of eEF2 in complex with moriniafungin or compound 1 was done essentially as described (25, 27). The antibiotics were solubilized in 100 mM Hepes, pH 7.2, and added to the concentrated eEF2 solution at a final concentration of 1-2 mg/ml prior to crystallization. The data were collected with synchrotron radiation to a maximum resolution of 2.9 Å for the eEF2-moriniafungin complex and 3.15 Å for the eEF2-compound 1 complex, and the data were processed with XDS (28) (Table 1). The structures of the two complexes were solved by molecular replacement with Molrep (29). The refined eEF2-sordarin structure (Protein Data Bank entry 1N0U [PDB] ) was divided into two search models containing residues 3-482 and 483-842, respectively. For the eEF2-moriniafungin complex the asymmetric unit contains two molecules of eEF2, whereas only one molecule is present for the eEF2-compound 1 complex. Following molecular replacement, the solutions were improved with rigid body refinement followed by positional refinement and grouped B-factor refinement to yield the initial model. The starting eEF2-moriniafungin model was completed by iterative cycles of rebuilding using O (30) and refinement with CNS (31). Tight noncrystallographic symmetry restraints were applied domain-wise between the two copies until Rfree was below 30%. Harmonic restraints were imposed on the main chain atoms. The structure of the eEF2-compound 1 was completed in similar manner, but without noncrystallographic symmetry restraints because there is only one complex in the asymmetric unit. The quality of the structures was inspected with PRO-CHECK (32), and domain movements were analyzed with Dyn-Dom (33). The coordinates and structure factors for both structures are deposited in the Protein Data Bank as entries 2NPF and 2E1R. All of the figures were created in PyMOL (34). Modeling of EF-G conformations based on structures of eEF2 in complex with sordarins or the conformation of eEF2 observed by cryo-EM (9) was performed in O. Diastereomers of compound 1 and moriniafungin were generated with an in-house distance geometry program based on published theory and algorithms (35, 36). A distance-dependent dielectric model of 2r was employed for all minimizations. The atoms of eEF2 were held fixed except for side chain within 5 Å of the modeled ligand, which were allowed to minimize in conjunction with the ligand and energy-minimized with the Merck molecular force field (37).


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  		TABLE 1
Statistics for data collection and refinement of the two structures

The values in parentheses are for the outer shell. The statistics for the Ramachandran plot are residues in most favored plus additionally favored regions/generously allowed regions/disallowed regions, excluding glycine and proline.

 


Figure 1
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  		FIGURE 1.
The chemical structures of the natural products sordarin and moriniafungin, as well as the semisynthetic analog compound 1.

 
Measurement of Ligand Binding to Yeast eEF2—The binding of ADP, ATP, mant-ADP, mant-ATP, and sordarin to eEF2 was measured in 0.5 cm x 0.5 cm quartz cuvettes by the quenching of intrinsic protein (Trp) fluorescence of the protein as a function of ligand concentration. In the case of the unmodified nucleotides and sordarin, the Trp quenching was a direct mechanism caused by the binding of the ligand to the eEF2 protein. The basis for Trp quenching induced by the mantnucleotides was due to fluorescence resonance energy transfer from the Trp to the mant-chromophore conjugated to the nucleotides. Triplicate measurements were performed over a concentration range of 0-50 µM (adenine nucleotides) or 0-100 µM (sordarin) in the presence of 0.5 µM eEF2 at 25 °C in an initial volume of 600 µl of buffer (20 mM Tris-HCl, 100 mM KCl, 1 mM dithiothreitol, 10% glycerol, 8 mM MgCl2, pH 7.6). The samples were excited at 295 nm (5-nm band pass), and the fluorescence emission intensity was measured at 340 nm (5-nm band pass) in a Cary Eclipse fluorescence spectrophotometer (Varian Canada Inc.) for titration experiments or in a PTI Alphascan-2 fluorescence spectrometer for wavelength scanning experiments. The fluorescence signal for the titration experiments was collected and averaged for 15 s for each measurement. A cuvette containing buffer only was also titrated with the corresponding ligand, and the signal from this sample was subtracted from the protein-containing samples. The fluorescence intensity measurements were also corrected for the dilution factor resulting from the titration (total dilution factor was less than 5%). The KD for sordarin or adenine nucleotides binding to eEF2 was determined using the following equation as part of the nonlinear fitting function of Origin 6.1 (OriginLab, Northampton, MA): {Delta}Fi/{Delta}Fmax = ([ligand] x Bmax)/(KD + [ligand]), where {Delta}Fi is the change in fluorescence intensity for each ligand concentration upon macromolecular association, {Delta}Fmax is the maximum change in fluorescence intensity at saturation of the ligand-binding site within eEF2, KD is the dissociation constant for the binding of each ligand with eEF2, and Bmax is the total eEF2 concentration (number of binding sites). The mant-derivatives of adenine nucleotides were excited at 350 nm, and the emission was scanned from 360 to 650 nm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
While possessing intrinsic antifungal activity that specifically targets eEF2, the parent natural product sordarin has limited spectrum of activity. In the current study we have determined the crystal structures of eEF2 in complex with two sordarin analogs (Fig. 1), both of which have improved potency and spectrum of fungal activity. Compound 1, an 11-chloro substituted analog of GM 237354 (22), contains a tetrahydrofuran ring with an exomethylene group fused at positions C-3' and C-4' of the sugar moiety of the sordarin molecule. Moriniafungin, a novel analog of sordarin isolated from the fungus Morinia pestalozzioides (24), contains a 2-hydroxysebacic acid residue linked to the C-3' of the sordarose moiety through a 1,3-dioxolan-4-one ring. To provide starting molecular models for fitting into the electron density for their respective complexes, we modeled both moriniafungin and compound 1 into the anti-fungal binding cleft in the eEF2-sordarin complex (25). The sordaricin aglycone core common to sordarin, moriniafungin, and compound 1 provided the initial docking. For both moriniafungin and compound 1, alternative possible conformers were studied while maintaining the initial core superposition and maximizing interactions between eEF2 and the additional substituents that differentiate these compounds from sordarin. This was trivially accomplished for compound 1 by fusing its tetrahydrofuran ring onto the terminal sugar of the crystallographic coordinates for sordarin. Moriniafungin was more problematic because of the flexible nature of the substituent as well as the unknown stereochemistry at two stereocenters. Ultimately, the two diastereomers of the 1,3-dioxolan-4-one in agreement with the NMR data, the C3'(R)/C-2''(S) (R/S) and C3'(S)/C-2''(R) (S/R) configurations (24), were modeled into the binding pocket. For each diastereomer, 150 conformations were generated, energy-minimized, rigidly superposed onto the sordarin core structure, and evaluated by complementarity to the binding cavity.

Upon positioning of the conformers for the two possible diastereomers of moriniafungin onto the sordarin template, it was apparent that the preponderance of the conformers for the octanylic acid moiety of the S/R diastereomer were in an equatorial relationship with the sordarose ring, whereas those of the R/S diastereomer were axially situated. Approximately 30 representative conformers for each diastereomer were energy-minimized within the context of the binding cavity (37). The lowest energy conformers of the S/R diastereomer that had the greatest interaction energy with eEF2 fit the octanylic acid moiety into a surface groove situated between the side chains of Gln795 and Val797 with the acid exposed to solvent. On the other hand, the lowest energy conformers of the R/S diastereomer filled the pore beneath the sugar moiety with the octanylic linker extending through the hydrophobic region created primarily by the side chains of Met796, Val488, and Gln490. Because such a location for the octanylic acid matched the electron density for the eEF2-moriniafungin complex, the R/S diastereomer was used for model building.


Figure 2
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  		FIGURE 2.
Four structures of eEF2. A, eEF2-sordarin (Protein Data Bank entry 1N0U). B, eEF2-compound 1. C, eEF2-moriniafungin. D, eEF2 bound to the 80 S ribosome as determined by cryo-EM (Protein Data Bank entry 1S1H). Binding of either moriniafungin or compound 1 causes a rotation of domains I-II of ~25°, giving the structures a more open conformation compared with eEF2-sordarin. The rotation angle between the eEF2-sordarin and the 80 S bound eEF2 structure is 56°. Domains I, G', and II are colored gray, domain III is blue, domain IV is red, domain V is green, and the sordarin/sordarin derivatives are yellow.

 
The structure of the eEF2-moriniafungin complex was determined to a maximum resolution of 2.9 Å (Table 1). There are only minor structural differences between the two copies in the asymmetric unit. In contrast to the structure of either eEF2-sordarin or ADP-ribosylated eEF2-sordarin, which both had p21212 symmetry (38), the space group for the eEF2-moriniafungin complex is p21. For the eEF2-compound 1 structure there is only one complex in the asymmetric unit and the space group p21212 with cell parameters related to those of eEF2-sordarin. Despite these differences in the crystal parameters for moriniafungin and compound 1, the two resulting structures are quite similar. The crystal packings within the crystals of eEF2-compound 1 and eEF2-moriniafungin do contain common elements, but many other packing interactions are not conserved between the two systems. This, combined with the similar unit cell parameters of eEF2-compound 1 and the original eEF2-sordarin structure, makes it likely that the novel conformation of eEF2 is not induced by similarities in the crystal packing for the two new structures. Instead, the conformational differences probably reflect an intrinsic conformation of eEF2 that is trapped by either of the two sordarin derivatives. However, we cannot completely exclude the effects of crystal packing. Compared with our previous structure of eEF2-sordarin, these two new structures have an eEF2 conformation in which domains III, IV, and V are rotated by 25° relative to domains I, G', and II (Fig. 2). The overall dimensions of this new conformation are 135 x 74 x 57 Å compared with 125 x 83 x 55 Å for the structure of eEF2-sordarin.

The electron densities for both moriniafungin and compound 1 allow unambiguous placement of all nonhydrogen atoms (Fig. 3, B and C). The overall location of moriniafungin and compound 1 bound to eEF2 is very close to that previously observed for sordarin. The tetracyclic diterpene is held in a pocket formed by residues from domains III, IV, and V of eEF2, whereas the substituents are recognized by residues in domains III and V only (Fig. 3, D-F). Interactions between the sordarins and eEF2 involve, in all three cases, both van der Waals' interactions and hydrogen bonds. Two hydrogen bonds are consistently formed from the diterpene carboxylic group and the hydroxylic group to main chain nitrogens in Glu524 and Val561 in eEF2, respectively. In both eEF2-sordarin and eEF2-moriniafungin, a third hydrogen bond is also formed between the pyran group and the oxaspiro group to the main chain nitrogen of Phe798. Sordarin and compound 1 have approximately the same interface area with eEF2, whereas moriniafungin has an interface more than 40% enlarged. This is caused by the insertion of the octanylic acid between Pro487, Val488, and Gln490 in domain III and Met796 in domain V. The terminal carboxyl group is tightly held by three hydrogen bonds to the main chain nitrogens of Gly779, Phe780, and Thr781 (Fig. 3E).

In both the eEF2-compound 1 and eEF2-moriniafungin structures, domains III-V are rotated by 25° relative to our original eEF2-sordarin structure (25). In both cases, this is apparently caused by a change in interactions between domains I and V. The two sordarin derivatives both cause the C terminus of the second helix in domain V, residues 780-787, to move slightly closer to domain III (Fig. 4). This causes a shift in interactions between domain V and the switch II/helix B region of domain I, allowing a partial closure of the domain I-V interface (Fig. 4). Extensive differences in this interface were earlier detected between the structures of apo-eEF2 and eEF2-sordarin (25), and the conformational flexibility of this interface is important for the function of EF-G (12).


Figure 3
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  		FIGURE 3.
The sordarins and their pockets. A, structures of eEF2 bound sordarin, moriniafungin, and compound 1 with gray carbon atoms, green chloride atoms, and red oxygen atoms. B and C, SigmaA weighted Fo - Fc omit electron density map contoured at 3.2{sigma} around moriniafungin (B) and around compound 1 (C). D-F, stereo views of the ligand-binding pocket in eEF2-sordarin (D), of the moriniafungin-binding pocket (E), and of the compound 1-binding pocket (F). The antibiotic-binding pockets are formed by residues from domains III (blue), domains IV (red), and domains V (green). Hydrogen bonds between eEF2 and the sordarins are shown with dashed lines.

 
Why do the two sordarin derivatives induce an altered conformation of eEF2 compared with the eEF2 conformation induced by sordarin? Both compound 1 and moriniafungin, but not sordarin, interact with Pro487 in the linker between domains II and III (Fig. 3, D-F). This may explain a distinct difference in main chain conformation observed at Pro487 and Val488 between the two conformations. In addition, a hydrogen bond between the side chain of Gln490 and the pyran moiety of sordarin is only observed in the eEF2-sordarin structure, where Gln490 also forms a hydrogen bond to the main chain oxygen of Pro487. In both the eEF2-moriniafungin and eEF2-compound 1 complexes, Gln490 is instead engaged in a hydrogen bond to Tyr521. In both structures, the insertion of the octanylic moiety in moriniafungin and the tetrahydrofuran ring with an exomethylene group in compound 1 between the two domains (Fig. 3, E and F) is likely to stabilize an altered domain III-V interface. In the eEF2-compound 1 complex, the absence of the hydrogen bond between the pyran ring and Phe798 found in both eEF2-sordarin and eEF2-moriniafungin (Fig. 3, D-F) might also contribute to the rotation of domain V toward domain III.


Figure 4
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  		FIGURE 4.
The interdomain area between domains I, III, and V. A, upon binding of the sordarin derivatives (moriniafungin or compound 1) the switch II/helix B regions move away from domain III and closer to domain V. The eEF2-sordarin complex is shown in gold, eEF2-moriniafungin is in blue, and eEF2-compound 1 is in gray. The three structures are superimposed on domain III of eEF2. B, domains III and V and the sordarin-binding site viewed from domain I. In contrast to sordarin (yellow), atoms from both moriniafungin (blue) and compound 1 (gray) penetrate into the space between domains III and V.

 
Except for the differences around the sordarin-binding pocket and the domain I-V interface, there are no major conformational differences within the six domains of eEF2. The residues Gln759-Gly762 are disordered in the eEF2-sordarin structure, but because of the closer approach of domain I and V in the eEF2-moriniafungin and eEF2-compound 1 structures, this loop becomes better ordered. We have modeled this loop in the eEF2-compound 1 complex and in one of two eEF2-moriniafungin complexes. Furthermore, the nucleotide-binding site contains a bound molecule of GDP. Density suggesting a Mg2+ ion is visible, but because of the low resolution, the ion cannot be reliably placed with a suitable stereochemistry in contrast to our structure of the ADP-ribosylated eEF2-sordarin complex (38). The new conformation of eEF2 is unlikely to be caused by the binding of GDP, because virtually no differences were observed in overall conformation between eEF2-sordarin without GDP and ADP-ribosylated eEF2-sordarin, which has GDP and Mg2+ bound as well.

Using fluorescence spectroscopy, we have determined the dissociation constant (KD) for ADP and ATP binding to yeast eEF2 (Fig. 5, A and B). Fluorescence emission spectra of eEF2 indicate that the addition of ADP and/or ATP quenches the intrinsic Trp fluorescence of the elongation factor in a manner analogous but not identical to the binding of the guanine nucleotides (38). The KD values for ADP binding to eEF2 were 0.94 ± 0.10 and 1.49 ± 0.21 µM in the absence and presence of GDPbetaS, respectively. Similarly, the KD values for ATP binding to eEF2 were 0.69 ± 0.09 and 0.68 µM in the absence and presence of GTP{gamma}S, respectively. Similar results were obtained using eEF2 from wheat germ (data not shown), indicating the general significance of these findings. These results indicate a lack of competition between the adenine and guanine nucleotides for binding with eEF2, suggesting that ADP and ATP bind to eEF2 at a site(s) that is distinct from the GDP/GTP-binding site located within domain I of the elongation factor. The binding of mant-nucleotide analogs could be monitored by both the enhancement of extrinsic analog fluorescence and the quenching of the intrinsic Trp fluorescence of eEF2. The fluorescence properties of the mant-linked analogs showed small but significant changes upon binding to eEF2; the emission maximum was shifted by 5 nm from 444 to 439 nm. Binding of the mant-nucleotides to eEF2 resulted in a partial quenching of the intrinsic Trp fluorescence of eEF2 (8 residues within yeast eEF2) and an increase in the extrinsic fluorescence of the mant-chromophore. Mant-ATP binding to eEF2 was prevented efficiently by a preincubation with either ATP or ADP but not GDP or GTP. These competition data show that the binding of the mant-nucleotides was uniquely restricted to the site of their corresponding unlabeled nucleotides. These results also suggest that two different sites exist on eEF2: one that is specific for the adenine nucleotides and a second that is specific for the guanine nucleotides.

Previously, we reported the binding of sordarin to yeast eEF2 as monitored by the quenching of the intrinsic Trp fluorescence of the protein (38). As illustrated in Fig. 5E, the addition of 100 µM ADP decreased the Trp fluorescence by 15% (trace b), and the addition of 100 µM sordarin further reduced fluorescence 10% (trace c). In contrast, when sordarin was added first, the intrinsic fluorescence of eEF2 was quenched by 25%, but the subsequent addition of ADP did not cause any further reduction in the intrinsic fluorescence of the elongation factor (Fig. 5F). These data indicate that sordarin and adenine nucleotides may bind to the same site within the eEF2 protein or that the sites are conformationally linked to each other.

The overall conformations of apo-eEF2 and EF-G-GDP are quite similar, indicating that the bacterial and the eukaryotic translocase have similar structural, functional, and conformational properties. There is also a considerable correlation between resistance to sordarin and to fusidic acid and to how the two antibiotics affect nucleotide exchange in yeast eEF2 (11). Binding of fusidic acid to EF-G and eEF2 requires ribosomes, and the affinity of eEF2 for sordarin is greatly enhanced by ribosomes (18). These similarities suggest that these two antibiotics target a similar functional state of eEF2, although they probably do not share binding sites. A suggestion that the conformation of eEF2 locked by sordarin onto the 80 S ribosome (9) is similar to the conformation of EF-G bound to the 70 S ribosome (39) in the presence of fusidic acid is supported by structural data. There is a strong resemblance between the cryo-EM reconstructions of the EF-G-GDP-70 S complex stabilized by fusidic acid and the eEF2-80S complex stabilized by the sordarin derivative GM 193663. In both cases, the ribosomes are in the same rotated conformation of the ribosome, and the overall locations of domains I, II, and IV are strikingly similar, whereas positions of domains III and V apparently differ somewhat for the 70 and 80 S ribosomes. However, the reliability of the eEF2 docking is probably significantly better than the docking of EF-G, because it was done in a 12 Å density map versus 18 Å for EF-G-70 S (39). A recent attempt to distinguish different populations of particles in the EF-G-70 S cryo-EM reconstruction (40) strongly indicates that the density previously assigned (especially domain III of EF-G) may be uncertain. In the fitting of eEF2, only two large rigid bodies derived from the crystal structure of eEF2-sordarin were used, whereas four rigid bodies were used for the EF-G, and the final fitted structure is rather different from the starting model derived from the crystal structure of EF-G GDP (41).


Figure 5
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  		FIGURE 5.
The binding of adenine nucleotides and sordarin ligands to eEF2. A, the quenching of the intrinsic Trp fluorescence of yeast eEF2 was determined from the titration of the protein with ADP in the presence (filled circle) or absence (open circle) of GDPbetaS. B, the quenching of ATP in the presence (filled triangle) or absence (open triangle) of GTP{gamma}S. Excitation was with 295-nm light, and the emission was measured at 340 nm with 5-nm bandwidths. C, evidence for fluorescence resonance energy transfer between eEF2 Trp residues and mant-ADP. Trace a is mant-ADP only, trace b is mant-ADP in the presence of eEF2, and trace c is eEF2 only. D, fluorescence resonance energy transfer between eEF2 and mant-ATP. Trace a is mant-ATP only, trace b is mant-ATP in the presence of eEF2, and trace c is eEF2 only. The samples were excited at 295 nm, and the emission was scanned from 305 to 540 nm, with 4-nm bandwidths for both excitation and emission. E, competition between sordarin and ADP for eEF2 trace a is eEF2 only, trace b is eEF2 in the presence of 100 µM ADP, and trace c is eEF2 where 100 µM ADP was added followed by 100 µM sordarin. F, dominant binding of sordarin to eEF2. Trace a is eEF2 only, trace b is eEF2 after the addition of 100 µM sordarin followed by trace c, 100 µM ADP. The excitation was at 295 nm, and the emission was scanned from 305 to 450 (E) or from 300 to 445 (F).

 
For these reasons, we investigated whether EF-G can adopt any of the previously known conformations of eEF2 in complex with various sordarin derivatives. We superimposed domains I-II, III, IV, and V of EF-G as individual bodies onto eEF2 in three different conformations, those of eEF2-sordarin (Fig. 2A), eEF2-moriniafungin (Fig. 2C), and eEF2 observed by cryo-EM (Fig. 2D). The G' domains of the two molecules are distinct and cannot be superimposed (25). Residues in the linkers between EF-G domains were modeled by homology to those of eEF2 as far as possible. The resulting models do not contain significant stereochemical clashes. We then mapped the location of 30 mutations in EF-G conferring resistance to fusidic acid onto the crystal structure of EF-G (41) (Protein Data Bank entry 1FNM [PDB] ), the EF-G-GDPNP complex (13) bound to the 70 S ribosome (Protein Data Bank entry 1PN6), the EF-G-GDP complex (39) stabilized onto the 70 S ribosome (Protein Data Bank entry 1JQM), and the three different models of EF-G were derived from eEF2-sordarin, eEF2-moriniafungin, and the cryo-EM conformation of eEF2 (9). The distances between C{alpha} positions of the fusidic acid resistance residues, excluding intradomain distances, were calculated in the six different cases, expecting the mean distance between these residues to be at the minimum in the conformation targeted by fusidic acid. The longest mean distance was obtained for Protein Data Bank entry 1JQM (33 Å); it was 26.4 Å for the EF-G crystal structure (Fig. 6A), whereas the shortest mean distance was 23.8 Å for the EF-G model derived from the cryo-EM conformation of eEF2 (Fig. 6B). The resistance mutations are located at the interface between domains I, III, and V of EF-G, so the decrease in the mean distance of our EF-G model derived from the cryo-EM conformation of eEF2 is caused by the closer approach of domain III (which contains one-half of the mutations) to both domains I and V (Fig. 6, A and B).


Figure 6
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  		FIGURE 6.
A conserved conformation of eEF2 and EF-G. A, fusidic acid related mutations mapped on the crystal structure of EF-G (Protein Data Bank entry 1FNM). The backbone of 30 fusidic acid resistance residues (41) is colored blue. B, mapping onto an EF-G model derived from the conformation of eEF2 observed by cryo-EM (see text for details). The closer approach of domain III to domain I in this model is illustrated by the distances between Leu457 and Gln117 (dashed lines). The distance is 16.5 Å in the crystal structure and 12.1 Å in the modeled structure. C, overlay of domains III (blue), IV (red), and V (green) of eEF2-sordarin and the equivalent domains of the T. thermophilus EF-G homolog (all domains gray) from Protein Data Bank entry 1WDT illustrating a highly conserved domain arrangement.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
There are now five different known conformations of eEF2, with four of them determined to high resolution by crystallography. These emphasize the organization of eEF2 into two structural blocks: an N-terminal region containing domains I, G', and II and a C-terminal region consisting of domains III, IV, and V (25). The C-terminal domains III, IV, and V can basically adopt two different conformations. In the crystal structures of eEF2 in complex with sordarin, moriniafungin, or compound 1, ligand binding causes domain III to rotate toward domain IV. During normal translation in the absence of sordarin, this conformation in the ribosomal environment probably occurs at least during the late phase of translocation, where eEF2 domain IV points into the 40 S decoding site (8, 9). This conformation is apparently not fungicide-dependent, because a similar domain arrangement was observed in the cryo-EM reconstruction of eEF2 stalled on mammalian 80 S ribosomes by a pseudoknot (42). In addition, a very similar arrangement of these domains is found in the structure of a Thermus thermophilus EF-G homolog in complex with GTP (Protein Data Bank entry 1WDT, 36% identity to EF-G), although it is unknown whether this molecule functions in translation (Fig. 6C). In domains III, IV, and V, 224 C{alpha} positions within residues 384-676 in the EF-G homolog and residues 490-821 of eEF2 can be superimposed with a root mean square deviation of 2.26 Å. The overall conformation of the EF-G homolog GTP complex is related to the cryo-EM conformation of eEF2 (9) by a rigid body rotation of 20° of domains III, IV, and V relative to domains I and II. The conformation of the EF-G homolog is related to the conformation of eEF2-moriniafungin by a rigid body rotation of 33° between the same two structural blocks.

The second conformation of domains III, IV, and V is observed in the structure of apo-eEF2 (25), and a very similar arrangement of these domains is found in the structure of the EF-G in complex with GDP (41). Furthermore, binding of the exotoxin A catalytic domain to eEF2 can induce a related third conformation, where domain III rotates by ~15° compared with apo-eEF2 (43).

It is clear from the available structures that in eEF2 and EF-G, domains III-V can move relative to domains I-II and that such movement can be caused by rather subtle changes (41, 44). The tetrahydrofuran ring with an exomethylene group in compound 1 that is fused to positions C-3' and C-4' of the sugar moiety of sordarin provides an example. This additional structural component causes domain III-V to rotate by 25° by inducing subtle rearrangements of domains III and V, which propagate to the domain I-II interface, thereby causing a substantial conformational change. Another example is EF-G, where an altered crystal packing caused a 10° reorientation of domains III-V relative to domains I-II. This furthermore caused domain III to become more highly ordered (41). In both eEF2 and EF-G, these changes lead to differences in contacts between switch II and domain III. This suggests that changes in switch II caused by GTP hydrolysis and release of inorganic phosphate can lead to substantial conformational changes in eEF2/EF-G, as has previously been observed for the translation factors EF-Tu (45) and eIF5B (46). However, the conformational properties of eEF2 and EF-G are probably mainly governed by the ribosome. Solution scattering studies of EF-G shows that its conformation in solution does not depend significantly on the nucleotide state of the factor (47).

Our structures suggest that even small changes introduced into sordarin can influence the conformational properties of eEF2 on the ribosome. This might explain differences in microbiological potency and spectrum as well as the detailed mechanism of inhibition. One study using unmodified sordarin suggests that sordarin blocks the elongation cycle prior to GTP hydrolysis and in a manner different to fusidic acid (48). A second study that utilized sordarin with the furan moiety replaced by a short alkyl ether, led to the suggestion that sordarin inhibits the release of eEF2 from the post-translocational ribosome in a manner similar to fusidic acid (11).

Massive rearrangements relative to the EF-G crystal structure of domains III-V are observed by cryo-EM in complexes of 70 S-EF-G stabilized by either GDPNP or fusidic acid (10, 13). The arrangement of the domains III-V in either of these reconstructions are apparently different from that in eEF2-sordarin complexes, but this might partially be caused by the rigid body approach used in these studies. Individual fitting of the small domains III and V into a low resolution density map is likely to be difficult, especially because the starting structure (that of EF-G-GDP) is rather distinct from the final fitted structure. In contrast, the fitting of these three domains of eEF2-sordarin as a single rigid body gave an excellent fit for the 80 S-eEF2 complex. Hence, in agreement with the very similar overall appearance of the cryo-EM reconstructions of eEF2-sordarin-80 S/EF-G-fusidic acid-70 S complexes, it is possible that the conformation of EF-G domains III-V on the ribosome are closer to that observed in the sordarin complexes of eEF2 than previously believed. In support of this, these three domains in EF-G can readily adopt the conformation they possess in eEF2 without stereochemical difficulties, and in this modeled conformation of EF-G, mutations that confer resistance to fusidic acid are closer than in any known structure of EF-G determined by crystallography or cryo-EM (Fig. 6, A and B). The resemblance of the GTP conformation of the EF-G homolog to the cryo-EM conformation of eEF2 (Fig. 6C) is a third indication that EF-G and eEF2 adopt similar conformations on the ribosome, which most likely change as a function of GTP hydrolysis, when bound to the ribosome.

Binding of ADP and ATP to a site distinct from the GDP/GTP-binding site has already been described for mammalian eEF2 (26), but the physiological function is uncertain. Based on detection of putative Walker A and B sequences, a binding site in the G' domain of eEF2 was suggested, but the suggested Walker A motif is not conserved in yeast eEF2, is not located at the N-terminal of an {alpha}-helix, and is not located next to the proposed Walker B motif in all known structures of yeast eEF2. The results presented in Fig. 5 confirm binding in a site distinct from the GDP/GTP-binding site and indicate that this is a conserved property of eukaryotic eEF2. None of the known structures of eEF2 contains any potential pockets on the surface besides the well characterized sites for binding of GDP/GTP in domain I and sordarin at the interface between domains III, IV, and V. Because sordarin can compete with ADP/ATP in a dominant manner (Fig. 5), it seems likely that the binding sites of sordarin and the adenylic nucleotides either overlap or that the conformation of eEF2 induced by binding of sordarin is incompatible with binding of these nucleotides. Binding of either ADP or ATP does significantly quench Trp fluorescence, indicating that their binding induces conformational changes in eEF2. The nature of this change may be related to that occurring upon binding of sordarin to eEF2. Based on these results, a binding site for ADP/ATP at the interface between domains III, IV, and V seems more likely than the previous suggestion for binding of these nucleotides to the G' domain (26).


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2NPF and 2E1R) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by Canadian Institutes of Health Research and Canadian Cystic Fibrosis Foundation. Back

2 Supported by the Danish Science Research Council, DANSYNC, the European Union Framework Program 5, and Merck Research Laboratories. To whom correspondence should be addressed: Dept. of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10C, DK-8000 Aarhus C, Denmark. Tel.: 45-89425024; Fax: 45-86123178; E-mail: gra{at}mb.au.dk.

3 The abbreviations used are: cryo-EM, cryoelectron microscopy; GDPbetaS, guanosine 5'-O-(2-thiodiphosphate); GTP{gamma}S, guanosine 5'-O-(3-thiotri phosphate). Back



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N. Demeshkina, G. Hirokawa, A. Kaji, and H. Kaji
Novel activity of eukaryotic translocase, eEF2: dissociation of the 80S ribosome into subunits with ATP but not with GTP
Nucleic Acids Res., July 9, 2007; 35(14): 4597 - 4607.
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