ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1998, p. 2279–2283 0066-4804/98/$04.0010 Copyright © 1998, American Society for Microbiology. All Rights Reserved.
Vol. 42, No. 9
Identification of Elongation Factor 2 as the Essential Protein Targeted by Sordarins in Candida albicans JUAN MANUEL DOMI´NGUEZ
AND
J. JULIO MARTI´N*
Departamento de Investigacio ´n, Glaxo Wellcome S.A. 28760-Tres Cantos, Madrid, Spain Received 26 January 1998/Returned for modification 22 April 1998/Accepted 24 June 1998
The target for sordarins in Candida albicans has been elucidated. Kinetic experiments of sordarin inhibition as well as displacement experiments showed that the formation of a sordarin-target complex follows a reversible mechanism. Binding of tritiated drug to the target is enhanced in the presence of ribosomes. Isolation of the target by classical protein purification methods has allowed us to identify it as elongation factor 2. This is in agreement with the nature of sordarin derivatives as specific inhibitors of the elongation cycle within protein synthesis in yeasts.
The appearance of resistance to current antifungal therapies in recent years has led to the need for new, effective drugs. In this regard, the finding of sordarins (11) as new antifungal drugs could be of potential value. One of the most attractive aspects of this family of compounds lies on its novel mode of action, inhibition of protein synthesis (6), an unusual feature in antifungal therapies. Taking into account the high degree of selectivity of sordarins, together with the conserved nature of the protein synthesis machinery within the eukaryotic kingdom, it was interesting to explore the precise mode of action of these drugs. Once it was established that the primary target was not the ribosome, we have studied the nature of the interaction between sordarin and its target on the pathogenic fungus Candida albicans, analyzing how this interaction is affected by the individual components of the fungal system. Furthermore, we have been able to purify the primary target for sordarin and conclusively identify it as elongation factor 2 (EF-2).
through Sephadex G-25, and the amount of drug bound to macromolecular components was determined by liquid scintillation counting. The binding constants for the C. albicans postribosomal supernatant (PRS) in the presence or absence of C. parapsilosis ribosomes were determined by equilibrium dialysis over 16 h at 30°C in a 150-ml final volume. (ii) Fractionation of PRS. Soluble factors from the C. albicans PRS were separated by the method described by Uritani and Miyazaki (33), except that Q-Sepharose and S-Sepharose columns were used and the last step (chromatography on hydroxyapatite) was omitted. (iii) Other methods. Preparation of C. albicans and C. parapsilosis cell-free translation systems, fractionation into ribosomes and PRS, and performance of the poly(U)-directed in vitro translation assay were done as described previously (6). ADP ribosylation of samples catalyzed with diphtheria toxin was carried out as described previously (30). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in 10% polyacrylamide gels as described previously (12). The protein concentration was determined as described by Bradford (4).
RESULTS In order to characterize the interaction between sordarin and its target, the reversible or irreversible nature of such an interaction was examined. This was initially done by kinetic experiments, in which we tested the effects of several sordarin concentrations on the rate of poly(U)-directed poly-[14C]Phe synthesis in a cell-free in vitro translation system from C. albicans. As shown in Fig. 1A, there was an immediate decrease in the rate of synthesis, and this decrease was related to the sordarin concentration. The rate of formation of the sordarintarget complex is therefore rapid. With the aim of confirming the reversibility of binding, displacement experiments were carried out by incubating the C. albicans cell-free system with [3H]sordarin, followed by the addition of excess unlabelled sordarin (Fig. 2). As can be seen, unlabelled sordarin was able to displace tritiated drug, and therefore, the reaction rates in Fig. 1A were analyzed and fitted to the following equilibrium binding equation: percent remaining activity 5 (100 z Ki/(Ki 1 [sordarin]), where Ki is the apparent inhibition constant, and [sordarin] is the concentration of sordarin. As shown in Fig. 1B, the experimental data closely fit the predicted values, with a Ki of 12 nM (5.9 ng/ml). With this in mind, isolation of the sordarin-target complex could be attempted only by fractionation methods under mild conditions that preserve the target conformation and its interaction with the sordarin molecule. Thus, it was possible to attempt purification of the target by conventional methods, taking advantage of the possibility of detecting the target by
MATERIALS AND METHODS Materials. The microorganisms used in the study (C. albicans 2005E and Candida parapsilosis 2372E) were obtained from the Glaxo Wellcome culture collection. Sephadex G-25 (PD-10 prepacked columns) and Q-Sepharose and S-Sepharose (1.6- by 10-cm prepacked columns) were from Pharmacia (Uppsala, Sweden). [14C]NAD (9.32 GBq/mmol) was from Amersham (Little Chalfont, United Kingdom). Sordarin (molecular weight, 492.6) was provided by the Bioprocessing Group, and [3H]sordarin (180 GBq/mmol) was synthesized by the Isotope Chemistry Group (both groups are at Glaxo Wellcome, Stevenage, United Kingdom). Anti-EF-1a and anti-EF-3 antibodies were a generous gift of M. F. Tuite. All other chemicals and reagents were of the highest quality available. All procedures were performed at 4°C unless stated otherwise. Methods. (i) Sordarin binding assays. Samples were incubated in the presence of 0.5 mg of [3H]sordarin (180 kBq/ml) per ml in a final volume of 500 ml at 25°C for 1 h. All samples were diluted in 30 mM HEPES-KOH (pH 7.4) containing 100 mM potassium acetate, 2 mM magnesium acetate, and 2 mM DL-dithiothreitol so that they had a standard protein concentration (10 mg/ml). Aliquots from chromatography eluates were previously mixed with 0.2 nmol of C. parapsilosis ribosomes. In the displacement experiments 100 mg of unlabelled sordarin per ml was added, and the sample was incubated for an additional 1 h. After the incubation period, unbound sordarin was removed in all cases by gel filtration
* Corresponding author. Mailing address: Departamento de Investigacio ´n, Glaxo Wellcome S.A., C/Severo Ochoa 2, 28760-Tres Cantos, Madrid, Spain. Phone: 34 91 8070301. Fax: 34 91 8070595. E-mail:
[email protected]. 2279
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FIG. 1. Kinetics of sordarin inhibition. (A) Poly(U)-directed in vitro translation was performed as described previously (6) with a cell-free system from C. albicans in the presence of several sordarin concentrations. The reaction was stopped by adding NaOH to 0.5 M each time, and the amount of poly-[14C]Phe synthesized was measured by trichloroacetic acid precipitation and liquid scintillation counting. The following symbols correspond to the indicated sordarin concentrations: E, 3.1 ng/ml; ■, 6.2 ng/ml; h, 12.5 ng/ml; Œ, 25 ng/ml; ‚, 50 ng/ml; ¿· , 100 ng/ml; F, control (without sordarin). (B) Fitting of experimental points to the theoretical curve corresponding to the equation for reversible inhibition (see text).
means of its ability to specifically bind to [3H]sordarin. Nevertheless, it is necessary to ascertain whether the sordarin-binding protein is the real functional target by proving a correlation between sordarin binding and inhibition of protein elongation. The data in Fig. 3A suggest that sordarin mostly bound to crude ribosomes from C. albicans, which appears to contradict previous results that suggested the nonribosomal nature of the sordarin target (6). To explain this, the following three hypotheses can be proposed: (i) The target is a ribosomal component. Previous results could be explained by assuming that resistant species contain a nonribosomal protein capable of replacing target function when this is inhibited. (ii) The target is a ribosomal component that interacts with a soluble factor which, according to its degree of affinity, might displace (resistance) or not displace (sensitivity) the sordarin-target interaction. (iii) The target is a soluble nonribosomal protein that remains attached to ribosomes after the centrifugation step. The sordarin-binding ability of this protein may be enhanced by its interaction with the ribosome. Of the three hypotheses, the first hypothesis seems to be the least feasible, because it assumes the existence of two genes encoding the same function, with the second gene being present specifically to prevent the antifungal effects of sordarin derivatives. Although this could be valid for genetically modified strains, it is unlikely to account for the situation in naturally occurring resistant species such as C. parapsilosis. On the other hand, the third hypothesis is in good agreement with the results presented in Fig. 3A, which indicate that KCl-washed ribosomes (devoid of any soluble factors) (32) have lost their ability to bind to sordarin, while the addition of PRS partially restores this ability. Moreover, ribosomes from sordarin-resistant species such as C. parapsilosis were unable to bind to [3H]sordarin, but when they were mixed with PRSs of sensitive species (C. albicans), binding of [3H]sordarin was considerably enhanced (Fig. 3B). Also, the inability of the ribosomes to bind to [3H] sordarin in the absence of soluble factors negates the second hypothesis since, according to that hypothesis, ribosomes from either a resistant or a sensitive species are expected to bind to [3H]sordarin. The resulting Kd value, obtained by equilibrium dialysis, for sordarin in C. albicans PRS was 3.11 mM, whereas in the mixture of C. albicans PRS and C. parapsilosis ribosomes the Kd value was 0.27 mM; i.e., C. parapsilosis ribosomes increase the affinity for sordarin 10 times. It is noteworthy that
in both cases a single class of binding sites was found (data not shown). On the other hand, no binding was detected when soluble factors from C. parapsilosis were mixed with washed ribosomes from C. albicans. These results lead us to conclude that the target is a soluble nonribosomal protein whose affinity toward sordarin is markedly increased in the presence of ribosomes, probably due to conformational arrangements in the target when it is interacting with the ribosome. From this conclusion good agreement between binding ability and resistance-sensitivity profiles can be stated, and therefore, binding to [3H]sordarin can be used to detect the sordarin target when trying to isolate it from the rest of the components of the C. albicans PRS. The isolation procedure was essentially the one followed by Uritani and Miyazaki (33). In the second chromatographic step (Fig. 4B) a single peak was able to bind sordarin. Western blot analysis with anti-EF-1a and anti-EF-3 antibodies revealed that none of these factors was present in this peak (data not shown). SDS-PAGE revealed that the major component of the
FIG. 2. Displacement of sordarin binding. C. albicans cell-free lysate was incubated in the presence of 0.5 mg of [3H]sordarin per ml at 25°C for 1 h. After adding buffer (A) or unlabelled sordarin in buffer to reach a final concentration of 100 mg/ml (B), the incubation was continued for an additional 1 h. (C) Lysate, 0.5 mg of [3H]sordarin per ml, and 100 mg of unlabelled sordarin per ml were incubated for 2 h. Subsequent to all the incubations unbound sordarin was removed by gel filtration, and the amount of bound drug, excluded as a component of a high-molecular-weight complex, was measured by liquid scintillation counting.
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FIG. 3. Sordarin binding to fractions from C. albicans (A) or C. parapsilosis (B). PRSs were separated from ribosomes by centrifugation at 100,000 3 g for 4 h; in the case of C. albicans, a portion of the ribosomes was subsequently washed with 0.5 M KCl. Afterwards, sordarin binding to each fraction was measured as described in Materials and Methods. (A) Fractions from C. albicans: 1, whole lysate; 2, PRS; 3, crude ribosomes; 4, KCl-washed ribosomes; 5, KCl-washed ribosomes plus PRS. (B) Fractions from C. parapsilosis: 1, whole lysate; 2, PRS; 3, crude ribosomes; 4, crude C. parapsilosis ribosomes plus C. albicans PRS.
peak was a protein of 98 kDa that was susceptible to ADP ribosylation by diphtheria toxin (Fig. 5), which exclusively recognizes EF-2 (3, 21). The elution profile of this protein (determined by ADP ribosylation [data not shown]) overlaps that of sordarin-binding activity. On the other hand, minor contaminants present are also radiolabelled with diphtheria toxin, while further chromatographic steps did not allow the separation of native EF-2 from its hydrolyzed contaminants. The bands from lane 9 in Fig. 5A were excised and sequenced. Figure 6 shows that the best sequence alignments in any case corresponded to Saccharomyces cerevisiae EF-2 fragments. From these results it is concluded that the major 98-kDa protein present in the sordarin-binding fraction is C. albicans EF-2. Minor contaminants coeluting with it seem to be derived from EF-2 proteolysis.
As shown in the previous section, the reversible interaction between sordarin and its target is greatly favored by the presence of ribosomes. It is well known that interaction with ribosomes promotes conformational changes on the three elongation factors (10) and that these changes lead to the appearance of a latent enzyme activity, i.e., GTPase for EF-1a and EF-2 and ATPase as well as GTPase for EF-3 (24–26, 28). On the ribosomal complex EF-2 increases its affinity for sordarin 10 times. This fact highlights the role of the ribosome as a modulator of the interaction between sordarin and its target, and taking into account the great diversity of macromolecular structures present in the ribosome (14, 15), this might provide
DISCUSSION The extremely high degree of selectivity of sordarins, a fact that favors their use as antifungal drugs, is a surprising event in light of its cellular target, one of the elongation factors involved in protein synthesis (6), because these factors are very well conserved proteins within the eukaryotic kingdom (16). So far, EF-3 is the only elongation factor reported to be present in yeasts but not in mammals (2, 31), and thus, it is commonly accepted as a suitable target for potential antifungal agents. Nevertheless, the selectivity can also come from subtle structural differences between other more conserved components of the protein synthesis machinery, differences that might be magnified by the complex interactions which are displayed within the elongation cycle (5, 17). Purification of the sordarin-binding protein from C. albicans PRS has led to the identification of EF-2 as the target of this new series of antifungal agents. All major bands present on SDS-PAGE were ADP ribosylated by diphtheria toxin, and their sequences showed homology with EF-2 from S. cerevisiae (22). The presence of bands with molecular weights lower than that of EF-2 is due to proteolysis of the factor, a fact that has been linked to the regulation of protein synthesis (7–9). Moreover, the existence of a specific protease for EF-2 has been described previously (29). With regard to this, it is noteworthy that, in this pool of C. albicans EF-2, all cleavage points are Asp-Pro bonds. So far this class of bond has not been described as a specific cleavage site for any protease, although selective hydrolysis of Asp-Pro bonds can be achieved under acidic conditions (13, 23).
FIG. 4. Purification of sordarin target from C. albicans PRS. The procedure of Uritani and Miyazaki (33) was essentially followed. Fractions were analyzed for their ability to bind to [3H]sordarin in the presence of C. parapsilosis ribosomes (J) as described in Materials and Methods. (A) Q-Sepharose chromatography at pH 7.5 (column of 1.6 by 10 cm, 0.8 ml/min, 5-ml fractions). (B) SSepharose chromatography at pH 7.5 (column of 1.6 by 10 cm, 0.6 ml/min, 2.5-ml fractions) of fractions from the chromatography described for panel A able to bind to [3H]sordarin. Abs, absorbance.
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FIG. 5. Electrophoresis analysis of S-Sepharose eluate. (A) SDS-PAGE of fractions from S-Sepharose eluate; lanes 1 and 10, molecular weight markers; lane 2, sample loaded onto the column; lanes 3 to 8, fractions unable to bind to [3H]sordarin; lane 9, fraction with maximum [3H]sordarin binding. (B) Fluorography of the sample corresponding to lane 9 after treatment with diphtheria toxin and [14C]NAD (lanes 2 and 3; lanes 1 and 4, molecular weight markers). Both panels are scanned images of the originals obtained by using Pharmacia Imagemaster; the images were furtherly processed as a tagged image file format with Microsoft Power Point.
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FIG. 6. Sequencing results for components from sordarin-binding peak in S-Sepharose eluate. Protein bands from lane 9 in Fig. 5A were excised and subjected to N-terminal sequencing. Sequence alignment was performed by the running BLAST program (1) on the Swiss-Prot database (Swiss Institute of Bioinformatics). The best scores were obtained with fragments of the sequence of EF-2 from S. cerevisiae (ScEF-2), as shown in the figure. aa, amino acids.
ity of a new antifungal target can be based solely on its primary structure. a clue to explain the selectivity of this kind of drug. Nevertheless, differences in single key residues at EF-2 itself may also account for the selectivity that has been found. The possibility of the sordarin interaction at the interface of the ribosome and the soluble factor can be ruled out because the PRS itself is able to bind to sordarin. EF-2, as well as its prokaryotic counterpart, EF-G, promotes translocation, i.e., displacement of nascent peptidyl-tRNA from the A site to the P site and movement of the ribosome along the mRNA; all of this is accompanied by a conformational change in the ribosome from the pretranslocational to the posttranslocational state (18). According to recent findings (27), GTP hydrolysis catalyzed by the elongation factor might provide the energy needed for the process. EF-2 is a highly conserved protein (85% homology and 66% identity between human EF-2 and S. cerevisiae EF-2) (22). It has been shown both for EF-G and for EF-2 from different sources that this protein is able to display different conformations depending on whether it is alone or interacting with GTP, GDP, or a ribosome in either the pre- or the posttranslocational state (19, 20). This conformational flexibility provides its biological properties and may be the reason that explains how such a conserved protein can be the primary target of such very selective antifungal drugs as sordarins. This fact might also lead to a revision of the idea that prediction of the apparent selectiv-
ACKNOWLEDGMENTS We thank J. M. Viana for technical assistance. The sequencing work performed by Edith Magnenat (Geneva Biomedical Research Institute, Geneva, Switzerland) is greatly acknowledged. Special thanks are given to M. F. Tuite (University of Kent, Canterbury, United Kingdom) for providing us with antibodies, to our colleagues at Glaxo Wellcome Research and Development (Stevenage, United Kingdom) for supplying sordarin, and to Michael S. Marriott for kind revision of this paper. REFERENCES 1. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410. 2. Belfield, G. P., N. J. Ross-Smith, and M. F. Tuite. 1995. Translation elongation factor-3 (EF-3): an evolving eukaryotic ribosomal-protein? J. Mol. Evol. 41:376–387. 3. Bodley, J. W., P. C. Dunlop, and B. G. van Ness. 1984. Diphthamide in elongation factor 2: ADP-ribosylation, purification and properties. Methods Enzymol. 106:378–387. 4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254. 5. Chakraburtty, K., and A. Kamath. 1988. Protein synthesis in yeast. Int. J. Biochem. 20:581–590. 6. Domı´nguez, J. M., V. A. Kelly, O. S. Kinsman, M. S. Marriott, F. Gomez de las Heras, and J. J. Martin. 1998. Sordarins: a new class of antifungals with selective inhibition of the protein synthesis elongation cycle in yeasts. Antimicrob. Agents Chemother. 42:2274-2278.
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