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Originally published In Press as doi:10.1074/jbc.M505951200 on October 28, 2005

J. Biol. Chem., Vol. 281, Issue 5, 2893-2900, February 3, 2006
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Enacyloxin IIa Pinpoints a Binding Pocket of Elongation Factor Tu for Development of Novel Antibiotics*Formula

Andrea Parmeggiani{ddagger}§1, Ivo M. Krab§2, Toshihiko Watanabe||, Rikke C. Nielsen{ddagger}, Caroline Dahlberg{ddagger}3, Jens Nyborg{ddagger}, and Poul Nissen{ddagger}4

From the {ddagger}Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10 C, DK-8000 Aarhus C, Denmark, §Laboratoire de Biophysique, Ecole Polytechnique, F-91128 Palaiseau Cedex, France, Department of Chemistry, Leiden University, P.O. Box 9502, NL-2300 RA Leiden, The Netherlands, and ||Department of Civil Engineering, Tohoku Institute of Technology, Sendai 982-8577, Japan

Received for publication, June 1, 2005 , and in revised form, July 25, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elongation factor (EF-) Tu·GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu·GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu·guanylyl iminodiphosphate (GDPNP)·enacyloxin IIa complex at 2.3 Å resolution presented here reveals the location of the antibiotic at the interface of domains 1 and 3. The binding site overlaps that of kirromycin, an antibiotic with a structure that is unrelated to enacyloxin IIa but that also inhibits EF-Tu·GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the higher binding affinity of the latter. EF-Tu·GDPNP·enacyloxin IIa shows a disordered effector region that in the Phe-tRNAPhe·EF-Tu (Thermus aquaticus)·GDPNP·enacyloxin IIa complex, solved at 3.1 Å resolution, is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elongation factor (EF)5 Tu is a GTPase that, as all members of this superfamily, cycles between the active GTP-bound and the inactive GDP-bound state. In bacterial protein synthesis EF-Tu is the carrier of aa-tRNA to the mRNA-programmed ribosome (for review see Refs. 1 and 2). The codon-anticodon interaction triggers the hydrolysis of the bound GTP, inducing the release of EF-Tu·GDP, the positioning of aa-tRNA into the ribosomal A-site, and peptide bond formation with the P-site-bound peptidyl-tRNA. EF-Tu folds in three distinct domains: the nucleotide-binding domain 1 (residues 1-199 in Escherichia coli (Ec) EF-Tu·GDP) shows the classical GTPase mixed {alpha}/beta Rossman fold, whereas domains 2 and 3 (residues 209-299 and 300-393, respectively) are beta-barrels. The association of the domains in EF-Tu·GDP with reduced interfaces and a central hole becomes more compact in EF-Tu·GTP with extensive interfaces and no hole (3-5). EF-Tu is the target of four structurally unrelated families of antibiotic inhibitors of protein synthesis (for review see Ref. 2), of which kirromycin and enacyloxin IIa hinder the release of EF-Tu·GDP from the ribosome after GTP hydrolysis, thus inhibiting its recycling and peptide bond formation. The high-resolution crystal structure of Thermus thermophilus (Tt) EF-Tu·GDP in complex with methylkirromycin (also called aurodox) showed that the antibiotic binds in the interface of domains 1 and 3, inducing a unique, compact EF-Tu-GTP-like conformation, which is the reason for its inhibition of the EF-Tu·GDP release (6). These conclusions have been further confirmed in studies of the Phe-tRNAPhe·EF-Tu·GDPNP·kirromycin.6 Specific differences between the enacyloxin IIa and kirromycin complexes with EF-Tu concern various aspects such as the electrophoretic migration on native gel, binding affinity, GTPase activity, different effects of mutations, etc. (7, 8).

In this work, we describe the structures of EF-Tu(Ec)·GDPNP·enacyloxinIIa t 2.3 Å and Phe-tRNAPhe·Thermus aquaticus (Ta) EF-Tu·GDPNP·enacyloxin IIa at 3.1 Å resolution. We have analyzed the antibiotic binding site and the induced structural and functional changes of EF-Tu in comparison with those of the GTP-like T. thermophilus EF-Tu·GDP·kirromycin complex. The similarities and differences in binding modes between the two antibiotics suggest new possibilities for structure-guided design of novel antibiotics with improved binding characteristics and possibly more selective action.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Crystallization and Data Collection—EF-Tu·GDPNP was formed by calf intestinal alkaline phosphatase (molecular biological degree, Roche Applied Science; 2 units/mg EF-Tu) cleavage of GDP from purified EF-Tu·GDP in 20 mM Tris-HCl (pH 8.0), 200 mM (NH4)2SO4, 2 mM dithiothreitol (DTT), 0.5 mM NaN3, and a 2.5-fold molar excess of GDPNP. Stock solutions (10 mM) of enacyloxin IIa in absolute ethanol at -81 °C were stable for at least 1 year. {epsilon}365 nm M-1 cm-1 = 91,000 in ethanol was used. For crystallization 60-180-µl solutions contained 4.0-4.5 mg/ml EF-Tu in 20 mM Tris-HCl buffer, pH 7.6, 20 mM NaCl, a 2-fold molar excess of GDPNP, 5-10 mM MgCl2, 2 mM DTT, 1% glycerol, 0.5 mM NaN3, and the antibiotic, added as the last component in a 1.2 molar ratio to EF-Tu. Higher molar excess of enacyloxin IIa over EF-Tu caused aggregation and precipitation of EF-Tu. After centrifugation, sitting drops of 2-5 µl of the clear supernatant mixed with a 0.4-0.5 volume equivalent from the reservoir (0.5 ml of 100 mM Tris-HCl, pH 7.4-7.6, 300-600 mM NaCl, 5-8% glycerol, and 20-23% polyethylene glycol 6000) were kept at 19 °C. Tetragonal crystals (space group P43212) with a faint yellowish color appeared within 24 h and grew as long rods to a maximum size of 1.5 x 0.5 x 0.3 mm within few days. The EF-Tu(Ta)·GDPNP·enacyloxin IIa·Phe-tRNAPhe (Saccharomyces cerevisiae) complex, obtained as described for the quaternary complex with kirromycin (9), was precipitated with (NH4)2SO4 and redissolved to 10 mg/ml in a crystallization buffer containing 1.25 M (NH4)2SO4, 10 mM MgCl2, 20 mM Tris-HCl, pH 7.2, 1 mM GDPNP, 0.5 mM DTT, and 50 µM enacyloxin IIa. Hanging drops (3-8 µl) were equilibrated at 4 °C against reservoir solutions containing 1 ml of 1.8 M (NH4)2SO4, pH 6.4-7.0. Monoclinic crystals (space group C2) appeared within a few days and grew to maximum dimensions of 0.6 x 0.4 x 0.2 mm.

EF-Tu·GDPNP·enacyloxin IIa crystals were mounted directly from mother liquor and flash-cooled in a 100 K cryostream (Oxford Cryosystems). X-ray crystallographic data extending to 2.3 Å resolution were collected from a single crystal at beam line I711 (MAX-Lab). Phe-tRNAPhe·EF-Tu·GDPNP·enacyloxin IIa crystals were flash-cooled in a cryoprotecting buffer consisting of 2.6 M (NH4)2SO4, 22% trehalose, 30 mM Tris-MES, pH 6.5, 10 mM MgCl2, 0.5 mM DTT, and 50 mM enacyloxin IIa, and data were collected at 100 K at beam line X13 (EMBL/DESY). Diffraction data were integrated and scaled with the HKL package (10), and structure factor amplitudes were calculated using Truncate of the CCP4 package (11). Table 1 shows the data statistics. The tRNA-bound complex (Table 1) suffered from strong anisotropy in the diffraction pattern, compromising data quality and subsequent refinement procedures.


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  		TABLE 1
Data collection and refinement statistics of EF-Tu·enacyloxin IIa (Enx) complexes

 
Structure Determination and Refinement—Structures were determined by molecular replacement using AMoRe (12) and search models derived from a kirromycin-bound complex (Protein Data Bank entry 1OB2).6 Two molecules of the EF-Tu·GDPNP·enacyloxin IIa complex related by a noncrystallographic 2-fold rotation axis (NCS) were located. Model phases were refined and extended by density modification using averaging, solvent flattening, and histogram matching in the program DM at 30-2.3 Å resolution (13). The resulting map was practically unbiased and showed clear density for enacyloxin IIa (Fig. 1B). Model building was performed in O (14), and an assigned C13-R, C14-S configuration for enacyloxin IIa provided the best fit. The model derived from the DM map was refined using crystallography NMR software (CNS) (15) initially at 5-2.3 Å resolution, enforcing strict NCS, and later at 30-2.3 Å resolution, using restrained NCS and bulk solvent correction. The {sigma}A-weighted 2Fo - Fc and Fo - Fc electron density maps were used in later rounds of model rebuilding. The final model comprised amino acid residues 9-40 and 55-393 for chain A and residues 6-41 and 57-393 for chain B as well as complete models of GDPNP·Mg2+ and enacyloxin IIa for both complexes. A total of 198 water molecules identified at hydrogen-bonding positions were included in the final model, and the final R-factor and Rfree factor of the model are 0.231 and 0.270, respectively, at 30-2.3 Å resolution (all data).

The EF-Tu·GDPNP·enacyloxin IIa·Phe-tRNAPhe structure contained three molecules in the asymmetric unit related by 3-fold NCS as observed also for the ternary complex without antibiotic (16) and with kirromycin.6 The complex was refined at 30-3.1 Å resolution as a single protomer with strict NCS throughout. The final model comprises a complete Phe-tRNAPhe molecule, EF-Tu residues 9-405, GDPNP·Mg2+, and enacyloxin IIa. The final R-factor and Rfree factor converged at 0.284 and 0.296, respectively, at 30-3.1 Å resolution (all data).

The figures were prepared using PyMol (33).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Enacyloxin IIa Binding Site on EF-Tu—Enacyloxin IIa (for its structure, see Fig. 2A) is active against Gram-positive and Gram-negative bacteria, slightly active against fungi, but inactive against yeasts (17-19). It stabilizes the EF-Tu·GTP complex (7) and enables EF-Tu·GDP to interact with aa-tRNA and ribosomes (8). Crystallographic analysis of EF-Tu·GDPNP·enacyloxin IIa locates the antibiotic at the "back" of the domain 1-3 interface (Fig. 1A), where a well ordered electron density delineates its boomerang-like structure (Fig. 1B). As a consequence of its insertion the interface is widened, and domain 1 is pushed upward and tilted forward (Fig. 1C). The antibiotic also reorients the side chain of several interface residues, breaking the hydrogen bonds Gln124 N{epsilon}2-Phe374 O and Tyr160 OH-Glu315 O{epsilon}. Thus, it binds by an induced-fit mechanism. At <3.8 Å it contacts 16 amino acids (7 from domain 1 and 9 from domain 3) via 6 hydrogen bonds and extensive van der Waals and hydrophobic interactions (Fig. 2, A, C, E, and G). A salt bridge links the carboxyl group of the hexane ring with the side chain N{zeta} of Lys313 (Fig. 2, A and C). It is noteworthy that all residues in the text are numbered according to EF-Tu(Ec), even when referring to EF-Tu(Tt)) or EF-Tu(Ta), with the homologous residue number in italics between parentheses. For more details of the enacyloxin IIa binding site, see Table 2.


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  		TABLE 2
Connections between enacyloxin IIa and its EF-Tu binding site and hydrogen bonds interconnecting residues at <3.8 Å from the antibiotic

A comparison with kirromycin is shown. All residues are numbered according to EF-Tu(Ec) even when referring to EF-Tu(Tt) as in the methylkirromycin complex. The homologous T. thermophilus residue number is shown in parentheses and italics. Bold letters indicate common amino acid residues.

 


Figure 1
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  		FIGURE 1.
Overview of the EF-Tu complex with enacyloxin IIa and comparison with other EF-Tu complexes and electron density map of enacyloxin-bound complex. A, "front" view of EF-Tu·GDPNP (GNP)·enacyloxin IIa. EF-Tu domains D1, D2, and D3 are shown in red, green, and blue, respectively. Helices in D1 are labeled with standard letter designations. The enacyloxin IIa (ENX) molecule is represented as a stick skeleton with the carbons shown in yellow, oxygens in red, carbamate nitrogen in blue, and chlorines in green. The dark green ball is the magnesium ion. B, unbiased electron density of enacyloxin IIa in the E. coli EF-Tu·GDPNP complex. The DM phase extension map at 2.3 Å resolution is displayed at 1.25 {sigma} around the refined model of enacyloxin IIa, showing all of the structural components of the antibiotic. Previously unassigned chiral centers are indicated by a blue asterisk. C and D show the same structure as in A, superimposed on other EF-Tu complexes represented by a semitransparent white "ghost." The orientations are chosen to visually maximize the differences in EF-Tu conformations. In all three panels the D2 and D3 of the two structures were superimposed by r.m.s.-minimized fitting of the backbone atoms of residues 207-246 and 251-393 (Ec) or their homologs 213-257 and 263-405 (Tt). C compares enacyloxin IIa-bound EF-Tu to the GDPNP-bound form of EF-Tu without antibiotic. The view is from the right side and slightly from below, compared with that in A. The insertion of enacyloxin IIa in the domain 1-3 interface at the "back" causes domain 1 to be pushed up and tilted slightly "forward" as indicated by the arrows. D shows that, compared with enacyloxin IIa, kirromycin causes in addition to the upward push and forward tilt of domain 1, an additional lateral displacement, roughly along the axis of helix A, by about half a turn.

 
Similarities to and Divergences from the Kirromycin Binding Site—In this work kirromycin is often used as synonym for methylkirromycin (Fig. 2B), because it is the prototype and most studied member of this family and displays the same effect on EF-Tu (20). From the three-dimensional model of the GTP-like EF-Tu(Tt)·GDP·kirromycin complex (6) the antibiotics binding site at the domain 1-3 interface shows striking similarities to that of the structurally unrelated enacyloxin IIa (Figs. 1D and 2, C and D) but also marked divergences. The surface area buried by enacyloxin IIa (~1200 Å2) is similar to that buried by kirromycin bound to EF-Tu·GDP or Phe-tRNAPhe·EF-Tu·GDPNP.6 The bulkier kirromycin widens the domain 1-3 interface of EF-Tu slightly more than enacyloxin IIa (by 15.4° versus 13.7°) (cf. Fig. 2, G and H). Of the 18 amino acids surrounding kirromycin at <3.8 Å, 9 (5 from domain 1 and 4 from domain 3) are also in close contact with enacyloxin IIa (Fig. 2, C and D; Table 2). Enacyloxin IIa makes hydrogen bonds with Arg123(124), Gln124(125), and Tyr160(161), as does kirromycin, although enacyloxin IIa only makes hydrogen bonds with the Arg123 main chain O, whereas kirromycin does so with the Arg123(124) side chain (Fig. 2, A-D). Enacyloxin IIa has two more hydrogen bonds to main chain residues, and kirromycin lacks the salt bridge to Lys313(325). In the enacyloxin IIa complex, the amino acid residues interacting with the antibiotic at <3.8 Å are interconnected with 4 hydrogen bonds and in the kirromycin complex with 8 hydrogen bonds. For more details of the two antibiotics binding sites, see Table 2.


Figure 2
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  		FIGURE 2.
Comparisons between enacyloxin IIa (left panels, yellow) and (1-methyl)-kirromycin (right panels, purple) binding sites showing their chemical structures (A and B), hydrogen bonds with EF-Tu (A-D), relationships with Gln-124(125) (E and F), and location in the interface gap between domains 2 and 3 with the hydrophobic pocket occupied by the tail of kirromycin (G and H). A and B, the structures are drawn schematically to facilitate comparison with C and D. The heads are on the left, and the "tails" on the right. Hydrogen bonds to EF-Tu structural elements are indicated by dotted lines and the salt bridge in A by larger dots. C and D, close-up of the antibiotics bound to EF-Tu in a "back" view compared with Fig. 1A. The antibiotics are shown as stick figures surrounded by transparent molecular surfaces. Hydrogen bonds are shown as green dotted lines and the salt-bridge in C as a red dotted line. Selected side chains and stretches of the EF-Tu backbone that make hydrogen bonds are shown as sticks on the schematic representation. Oxygen atoms are red, nitrogens are blue, and the chlorines in enacyloxin IIa are bright green. Note that the heads of the two antibiotics point in opposite directions; that of enacyloxin points toward the viewer, whereas that of kirromycin points away (into the EF-Tu). Also note that the tail of kirromycin points away into the interior of EF-Tu. This latter point is illustrated more clearly in H. In E-H, the EF-Tu is shown as a molecular surface with selected elements identified by labels. The color scheme is again red for domain 1 and blue for domain 3. In E and F, the complex relationships between kirromycin and Gln124(125) (green area) with several contacts is compared with that of enacyloxin IIa. For a close comparison, E and F also outline the overlap with kirromycin and enacyloxin IIa, respectively. The green area in G and H is the hydrophobic surface of atoms within4 Å of the kirromycin tail (or the equivalent atoms in G). Clearly the tail of enacyloxin IIa borders this pocket, whereas that of kirromycin occupies it. Note that in C and D, domains 1 are in the exact same position, whereas domains 3 are pushed down and to the left as a result of the greater bulk of kirromycin. This larger gap between the domains can be recognized in H on the left side (cf. G).

 


Figure 3
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  		FIGURE 3.
Labile binding of enacyloxin IIa to EF-Tu as compared with kirromycin. Poly(Phe) synthesis was performed with EF-Tu ({square}, {blacksquare}), EF-Tu·enacyloxin IIa ({triangleup}, {blacktriangleup}), or EF-Tu·kirromycin ({circ}, bullet) after passing the preformed EF-Tu·antibiotic complex through a 7-cm-long Sephadex G50 column, either alone (open symbols) or as its complex with Phe-tRNAPhe (filled symbols). A 200-µl eluate containing EF-Tu was collected, and 45 µl of the solution was added to 15 µl of the mix (preincubated for 15 min at 30 °C) containing the other components necessary to reconstitute a poly(Phe) synthesis system, with final conditions of 25 mM Hepes, pH 7.6, 70 mM NH4Cl, 7 mM MgCl2, 7 mM beta-mercaptoethanol, 1.5 mM ATP, 0.5 mM GTP, 3.8 mM phosphoenolpyruvate, 30 µg/ml pyruvate kinase, 1.5 µM ribosomes, 50 µg/ml poly(U), 0.8 µM elongation factor G, 0.05 µM EF-Ts, 3 µM tRNAPhe, 20 µM [14C]Phe (160 dpm/pmol), and 25 nM Phe-tRNA synthetase. At the indicated times during incubation at 30 °C, 10-µl samples were withdrawn, spotted on glass fiber filters (Whatman GF/A), and quenched in ice-cold 10% trichloroacetic acid, and the poly(Phe) synthesized was determined. This experiment was repeated three times with similar results.

 
The hydrophobic contacts of enacyloxin IIa with domain 1 residues show important differences from those of kirromycin. The enacyloxin IIa polyenic chain is loosely bound at a patch with Gln159, Tyr160, Asp161, and Arg123 on the one side and Leu120, Arg123, and Gln124 on the other side, steering clear of the side chain of the latter (Fig. 2C). On the other hand, the chain of kirromycin interacts closely with both Gln124(125) and Leu120(121), whereas the central pentose tightly fills the groove at the domain 1-3 interface, because of its hydrogen bonds with Arg123(124) (Fig. 2, D, F, and H). The "head" of enacyloxin IIa touches the tip of the loop formed by domain 3 residues 313-316, whereas that of kirromycin points the other way, wrapping around Leu120(121) and Tyr160(161) and displacing the Glu117(118) side chain. Furthermore, the "tail" of kirromycin fills the hydrophobic pocket between Gln124(125) and Ile92(93), whereas enacyloxin IIa passes along the outside of the cavity (Fig. 2, G and H). This is in line with the weaker binding of enacyloxin IIa, which is released from EF-Tu on short gel filtration columns, whereas kirromycin is not (see Ref. 7 and the representative experiment in Fig. 3). With enacyloxin IIa the presence of aa-tRNA accelerates this effect. This is important because in the cell most EF-Tu·GTP is bound to aa-tRNA. The results of this experiments demonstrates that the binding affinity of enacyloxin IIa for EF-Tu is much lower that that of kirromycin, as expected from the properties of their binding to EF-Tu. Under the chosen conditions a 50% release of enacyloxin IIa from EF-Tu in ~20 min can be estimated, whereas that of kirromycin is negligible. The determination of the dissociation constant of the two EF-Tu·antibiotic complexes was not carried out because of the very low water solubility of enacyloxin IIa and kirromycin and their tendency to induce aggregation of EF-Tu. The domain 3 residues contacting both antibiotics form a more or less flat surface, with bumps that are in both cases tightly complementary to the antibiotic. Arg373(385) "embraces" the active core (Fig. 2, C, D, G, and H).

Overall and Specific Changes in EF-Tu Domains and Phe-tRNAPhe Complex—In EF-Tu·GDPNP· enacyloxin IIa the overall domain arrangement resembles that of EF-Tu·GDPNP in the absence of the antibiotic (Fig. 1C). In the enacyloxin IIa complex the C terminus of helix C (residues 89-93) is slightly pushed up, whereas helix B is pulled roughly along its axis toward the tail of the antibiotic. As a consequence the effector region, residues 41-65, that in EF-Tu·GDPNP contacts this helix is destabilized and changes conformation. Its N-terminal part (residues 41-50) shows no electron density, although in the residual part (Switch 1 region) the weak electron density reveals an unwound a-helix A'' collapsed into domain 2 (Fig. 1A). Arg58(59) and Thr61(62), two conserved residues important for GTPase activity and interaction with the ribosome (21, 22), are moved away by ~15 Å toward the vacant binding pocket for the aminoacyl residue of tRNA. Fig. 1D illustrates the superimposition of EF-Tu·GDPNP·enacyloxin IIa and EF-Tu·GDP·-kirromycin. Also the kirromycin complex shows a disordered effector region (6). For the changes in the Switch 1 region of the two antibiotic complexes, see also Fig. 6. Most important, on binding of Phe-tRNAPhe to EF-Tu·GDPNP·enacyloxin IIa the disordered effector region is restructured by the interaction of the {alpha}-helix A'' with tRNA (Fig. 4). The acceptor stem and part of the CCA-end of tRNA are tugged by the effector region to follow the displacement of domain 1 induced by the antibiotic binding, whereas the remaining tRNA regions follow domains 2 and 3. As a result, the acceptor stem twists relative to the T-stem and displays a unique conformation intermediary between free tRNA and tRNA in the ternary complex (Fig. 4). The phenylalanyl group has a reduced electronic density, indicating a defective accommodation (Fig. 5) that is reflected in the decreased protection by EF-Tu·GTP against the spontaneous deacylation of Phe-tRNAPhe, which is more pronounced than in the case of kirromycin (see supplemental Fig. 1). The Switch 2 region of the enacyloxin IIa complex shows no marked changes. The side chain of His84(85), a residue important for the GTPase activity dependent on EF-Tu (23-25), points away from the {gamma}-phosphate of the nucleotide similarly to the native conformation (Fig. 6). In contrast, in the kirromycin complex it is turned toward the phosphate groups of GDP. Enacyloxin IIa enhances the intrinsic GTPase activity of EF-Tu but much more weakly than kirromycin (Fig. 7; note the difference in scale between panels A and B). Interestingly, with enacyloxin IIa the different ions show similar concentration dependence, the "soft" Formula being however the strongest stimulator, whereas with kirromycin EF-Tu displays a GTPase increase as a function not only of the increasing concentration of "hard" monovalent ions but also of their charge density.


Figure 4
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  		FIGURE 4.
Phe-tRNA from the quaternary complex (QC) with EF-Tu·GDPNP·enacyloxin IIa as compared with the native ternary complex (TC) and free tRNA. The acceptor stem of tRNAPhe from the quaternary complex is represented in yellow, from the native ternary complex Phe-tRNAPhe·EF-Tu·GDPNP in blue, and from free tRNAPhe in dark gray. Note the intact effector region with helix A".

 


Figure 5
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  		FIGURE 5.
The final 2Fo - Fc electron density map displayed at 1.0 {sigma} of the EF-Tu(Ta)·GDPNP·enacyloxin IIa complex with Phe-tRNAPhe. Note the following: (i) the Switch 1 region over the tRNA acceptor stem, showing density for the main chain conformation as in the native ternary complex; (ii) the 3'-phenylalanine group with poor definition in the electron density map, which is likely caused by disorder.

 


Figure 6
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  		FIGURE 6.
Comparison of the orientation of His84(85) in the Switch 2 region (loop L4 and helix B) in the different complexes. Superimposition was carried out by r.m.s. minimization of structurally equivalent domain 1 backbone atoms. Letters designate helices. GDP, GNP (GDPNP), ENX (enacyloxin IIa), and KIR (kirromycin) label the His84(85) side chains of EF-Tu·GDP (blue), EF-Tu·GDPNP (green), EF-Tu·GDPNP·enacyloxin IIa (yellow), and EF-Tu·GDP·methylkirromycin (purple), respectively. Domain 1 is shown in a schematic representation, and the His84(85) side chain, together with the bound nucleotide and antibiotics, is shown in stick representation with Mg2+ shown as a sphere. Note the heads of enacyloxin IIa and kirromycin pointing in opposite directions, at right angles to the body of the antibiotics. Note also the different conformations of the Switch 1 region in the enacyloxin IIa complex, and in the GDPNP and GDP complexes in the absence of the antibiotic. This region is disordered in the EF-Tu·GDP·methylkirromycin complex.

 


Figure 7
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  		FIGURE 7.
The intrinsic GTPase activity of EF-Tu in the presence of enacyloxin IIa (A) or kirromycin (B) as a function of the concentration and kind of monovalent cations. EF-Tu·[{gamma}-32P]GTP was preformed by incubating EF-Tu·GDP (200-400 pmol) for 20 min at 30 °C in a 65-µl solution of 50 mM Tris-HCl, pH 7.5, an excess (5-20-fold) of [{gamma}-32P]GTP (specific activity, 500-4000 cpm/pmol) over EF-Tu, 10 mM MgCl2, 2 mM phosphoenolpyruvate, 50 µg/ml pyruvate kinase, and 1 mM ATP. Thereafter, LiCl ({circ}), NaCl ({square}), KCl ({triangleup}), or NH4Cl ({diamond}), to a final concentration as indicated plus 50 µM enacyloxin IIa (A) or kirromycin (B), was added to aliquots of the reaction mixture kept at 0 °C. GTP hydrolysis was started by a temperature shift to 37 °C. Several samples were withdrawn in a period of time (15-60 min) in which the GTPase rate was linear, and the GTPase activity was determined for each salt concentration kinetically as 32Pi liberation after charcoal treatment (32). The background of the intrinsic GTPase activity of EF-Tu in the absence of antibiotics was subtracted. Data points represent an average over three experiments, and error bars indicate S.D.

 


Figure 8
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  		FIGURE 8.
Structural divergences between enacyloxin IIa and kirromycin and some specific effects. A and B show the structural background of the effect of the A375V mutation on the sensitivity of EF-Tu toward enacyloxin IIa and kirromycin. The panels show close-up views in the same orientation of the enacyloxin IIa (A) and the kirromycin (B) complex of EF-Tu. Ala375 has been substituted in silico by Val (gray). The transparent van der Waals sphere around the beta-carbon indicates the space occupied by an Ala side chain. The stippled surface indicates the space occupied by Val. Clearly, the tail of enacyloxin IIa is not hindered, but rather it makes a more extensive hydrophobic contact with the Val375 side chain. In contrast, there is a steric clash for the kirromycin tail entering the hydrophobic pocket; the transparent van der Waals surface (purple) around the tail intersects with the Val375(387) van der Waals envelope (glossed). C, selective differences in the tail moiety of the overlap between enacyloxin IIa (yellow) and kirromycin (pink), likely to affect the binding affinity and activity of the two antibiotics. The active core area of kirromycin is circled.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This work describes for the first time the structure of the EF-Tu·enacyloxin IIa complex, defining the antibiotic binding site on EF-Tu, which in the domains 1-3 interface overlaps that of kirromycin. There is strong evidence that despite a number of functional differences both compounds inhibit protein synthesis by inducing a unique GTP-like conformation of EF-Tu·GDP (8). Accordingly, the overall structures of EF-Tu·GDPNP·enacyloxin IIa and EF-Tu·GDP· kirromycin, and the binding sites of the two antibiotics share marked similarities. Nonetheless there are important, specific divergences, such as the less complex contact of enacyloxin IIa with Gln124(125) and the behavior of the short tail of enacyloxin IIa, which borders an empty hydrophobic pocket, whereas the longer tail of kirromycin fits into this pocket. This, we believe, represents the main reason for the lower binding affinity of enacyloxin IIa to EF-Tu. Related to this aspect is the differential effect caused by substituting the bordering Ala375(387) with the bulkier Val that fills this pocket. EF-Tu becomes 3-fold more sensitive to enacyloxin IIa (8) but ~300 times more resistant to kirromycin (26). The insertion of the Val375 side chain stabilizes the short tail of enacyloxin IIa by extending the contacting hydrophobic surface (Fig. 8A), whereas it inhibits the fitting of the longer tail of kirromycin by steric hindrance (Fig. 8B). Fig. 8C illustrates the overlap of enacyloxin IIa and kirromycin, underlining their overall structural divergences and similarities; it also indicates the active core of kirromycin containing the tail half-molecule as deduced from the functional analysis of fragments and chemical modifications of the antibiotic (27-29). Our observations hint at new approaches for a structure-guided antibiotic design on the basis of the hybridization of enacyloxin and kirromycin structural components, with the aim of obtaining novel EF-Tu binding compounds with specific properties. From our results, combination of the head moiety of enacyloxin IIa with the tail moiety of kirromycin should increase the binding affinity of the resulting compound. The binding efficiency of an antibiotic can affect the degree of the antibacterial effect and reduce the onset of resistance by microorganisms. One can extend this approach by creating hybrids between structural parts of the two antibiotics and other cyclic compounds (e.g. sugars, lactones, macrolides, etc.). By taking into account the common properties of the two antibiotics binding sites, one could combine any structural moieties making hydrogen bonds with amino acids around the head of the antibiotic, e.g. Tyr160(161) with compounds bonding residues around the tail, such as Glu124(125) and Lys313(325).

Another interesting result of this work is the ability of aa-tRNA to revert the disorder of the effector loop caused by the binding of the antibiotic. This effect is associated with a unique distortion of the acceptor stem of tRNA. This, however, does not impair the EF-Tu-mediated binding of aa-tRNA to the programmed ribosome, which in the presence of enacyloxin IIa is even improved (7). A possible explanation for this effect can by derived from the observation that the similarly distorted tRNA bound to EF-Tu·GDPNP·kirromycin shares common features with the conformation of the ribosome-bound EF-Tu·GDPNP·kirromycin complex.6 Concerning the high mobility of the effector region, a plausible mechanism can be found in the distortion of helices B and C induced by the accommodation of the antibiotic in the domain-1-3 interface. The signal arising from the binding of the antibiotic can thus be transferred to other areas of EF-Tu.

It is not clear why EF-Tu·enacyloxin IIa shows a lower intrinsic GTPase activity than EF-Tu·kirromycin. Site-directed mutagenesis did not prove the involvement of His84 side chain in a nucleophilic attack on the {gamma}-phosphate; however, it supported an indirect key role of this residue in both the intrinsic and the very fast GTP hydrolysis evoked by programmed ribosomes (23, 24). To date the most convincing hypothesis about the mechanism of the low intrinsic EF-Tu GTPase activity is "substrate-assisted" GTP hydrolysis (30). The level of intrinsic GTPase activity of EF-Tu likely depends on selective local pH effects influencing the stability of the {gamma}-phosphate, which could be differently affected by the two antibiotics and which may also relate to the differential influence of cations on this activity.

In conclusion, the structural similarities and differences between enacyloxin IIa and kirromycin will not only explain central features of their action but also evoke selective approaches for obtaining novel inhibitors of protein synthesis, a proven target and a crucial aspect for society, taking into account the general increase in resistance toward antibiotics.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2BVN and 1OB5) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by an Ole Rømer stipend, the Dansync Project of the Danish Natural Science Research Council, European Union Contract QLK2-CT-2002-00892, Grant 21-03-0214 from the Danish Natural Science Research Councils Centre for Structural Biology, and the European Molecular Biology Organization Young Investigator program. 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental material. Back

2 Present address: Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Rd., Ag. Sci. 218, Honolulu, HI 96822-2321. Back

3 Present address: Dept. of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195-7350. Back

1 To whom correspondence may be addressed: Dept. of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10 C, DK-8000 Aarhus C, Denmark. Tel.: 45-8942-5258; Fax: 45-8612-3178; E-mail: andrea{at}bioxray.dk.

4 To whom correspondence may be addressed. Tel.: 45-8942-5025; Fax: 45 8612 3178; E-mail: pn{at}mb.au.dk.

5 The abbreviations used are: EF, elongation factor; aa-tRNA, aminoacyl-tRNA; D1, D2, and D3, EF-Tu domains 1, 2, and 3, respectively; GDPNP, guanylyl iminodiphosphate; Ec, Escherichia coli; Tt, Thermus thermophilus; Ta, Thermus aquaticus; DTT, dithiothreitol; r.m.s., root mean square; MES, 4-morpholineethanesulfonic acid. Back

6 R. C. Nielsen, O. Kristensen, and P. Nissen, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank Ray Brown for many helpful discussions. We are grateful for beamtime at beamline I711 (MAX lab) and beamline X13 (DESY/EMBL).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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L. N. Olsthoorn-Tieleman, R.-J. T. S. Palstra, G. P. van Wezel, M. J. Bibb, and C. W. A. Pleij
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