FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2042211679> ?p ?o ?g. }

• W2042211679 endingPage "31407" @default.
• W2042211679 startingPage "31402" @default.
• W2042211679 abstract "Candida albicans EF-2 binds sordarin to a single class of binding sites with K d = 1.26 µm. Equimolar mixtures of EF-2 and ribosomes, in the presence of a non-hydrolyzable GTP analog, reveal two classes of high affinity sordarin binding sites with K d = 0.7 and 41.5 nm, probably due to the existence of two ribosome populations. Photoaffinity labeling of C. albicans EF-2 in the absence of ribosomes has been performed with [14C]GM258383, a photoactivatable sordarin derivative. Labeling is saturable and can be considered specific, because it can be prevented with another sordarin analog. The fragment Gln224–Lys232 has been identified as the modified peptide within the EF-2 sequence, Lys228 being the residue to which the photoprobe was linked. This fragment is included within the G“-subdomain of EF-2. These results are discussed in the light of the high sordarin specificity toward fungal systems. Candida albicans EF-2 binds sordarin to a single class of binding sites with K d = 1.26 µm. Equimolar mixtures of EF-2 and ribosomes, in the presence of a non-hydrolyzable GTP analog, reveal two classes of high affinity sordarin binding sites with K d = 0.7 and 41.5 nm, probably due to the existence of two ribosome populations. Photoaffinity labeling of C. albicans EF-2 in the absence of ribosomes has been performed with [14C]GM258383, a photoactivatable sordarin derivative. Labeling is saturable and can be considered specific, because it can be prevented with another sordarin analog. The fragment Gln224–Lys232 has been identified as the modified peptide within the EF-2 sequence, Lys228 being the residue to which the photoprobe was linked. This fragment is included within the G“-subdomain of EF-2. These results are discussed in the light of the high sordarin specificity toward fungal systems. elongation factor 2 elongation factor G β:γ-imidoguanosine 5′-triphosphate matrix-assisted laser desorption/ionization mass spectroscopy high performance liquid chromatography The natural product sordarin and its semisynthetic derivatives constitute a selective class of inhibitors of protein synthesis in fungi. Their potential as systemic antifungal agents has been evidenced by their broad spectrum and in vivo therapeutic efficiency (1Gargallo-Viola D. Curr. Opin. AntiInfect. Invest. Drugs. 1999; 1: 297-305Google Scholar, 2Avilés P. Falcoz C. San Román R. Gargallo-Viola D. Antimicrob. Agents Chemother. 2000; 44: 2333-2340Crossref PubMed Scopus (27) Google Scholar; see Ref. 3Odds F.C. Exp. Opin. Ther. Patents. 2001; 11: 283-294Crossref Scopus (39) Google Scholar for a recent review). Among all the processes included in protein synthesis, ribosomal translocation is the step impaired by sordarin (4Domı́nguez J.M. Gómez-Lorenzo M.G. Martı́n J.J. J. Biol. Chem. 1999; 274: 22423-22427Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). This molecule binds to EF-21 itself, although such binding is greatly favored by the presence of ribosomes (5Domı́nguez J.M. Martı́n J.J. Antimicrob. Agents Chemother. 1998; 42: 2279-2283Crossref PubMed Google Scholar). On the other hand, mutations leading to sordarin resistance have been found both in EF-2 and in the yeast ribosomal protein rpP0 (6Justice M.C. Hsu M.J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 7Gómez-Lorenzo M.G. Garcı́a-Bustos J.F. J. Biol. Chem. 1998; 273: 25041-25044Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Hence, the EF-2·ribosome complex is proposed as the functional target of sordarin antifungals. Translocation has been thoroughly studied in prokaryotes and a good picture of the global mechanism at molecular level is starting to emerge. Key aspects in such progress have been the elucidation of ribosome structure at good resolution (8Ban N. Nissen P. Hansen J. Capel M. Moore P.B. Steitz T.A. Science. 1999; 400: 841-847Google Scholar) and detailed crystallographic studies of EF-G, the prokaryotic homolog of EF-2 (9Czworkowski J. Wang J. Steitz T.A. Moore P.B. EMBO J. 1994; 13: 3661-3668Crossref PubMed Scopus (360) Google Scholar, 10Aevarsson A. Brazhnikov E. Garber M. Zheltonosova J. Chirgadze Y. Al-Karadaghi S. Svensson L.A. Liljas A. EMBO J. 1994; 13: 3669-3677Crossref PubMed Scopus (333) Google Scholar). Also, visualization of the ribosome·EF-G complex by cryo-electron microscopy (11Agrawal R.K. Penczek P. Grassucci R.A. Frank J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6134-6138Crossref PubMed Scopus (306) Google Scholar), as well as other functional studies (12Rodnina M.V. Savelsbergh A. Katunin V.I. Wintermeyer W. Nature. 1997; 385: 37-41Crossref PubMed Scopus (392) Google Scholar, 13Peske F. Matassova N.B. Savelsbergh A. Rodnina M.V. Wintermeyer W. Mol. Cell. 2000; 6: 501-505Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), have led to a better understanding of the process (see Ref. 14Green R. Curr. Biol. 2000; 10: R369-R373Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar for a review). EF-G, the protein that promotes ribosomal translocation in prokaryotes, is a tadpole-like molecule organized into five domains. The globular domain I is responsible for GTP binding and hydrolysis. It contains several structural motifs characteristic of the G-protein superfamily (indeed, it is referred to as “G-domain”) plus an extra insert called the G′-subdomain. The role of the latter remains unknown, although it has been suggested to act as a nucleotide-exchange factor (9Czworkowski J. Wang J. Steitz T.A. Moore P.B. EMBO J. 1994; 13: 3661-3668Crossref PubMed Scopus (360) Google Scholar). Interaction of the G-domain with the ribosome triggers GTP hydrolysis, and the energy released is transformed into mechanical movement. Eventually, domain IV (a fibrous domain at the other end of the molecule) moves away, stretching the EF-G shape and literally pushing the newly formed peptidyl-tRNA from the A to the P ribosomal site (15Agrawal R.K. Heagle A.B. Penczek P. Grassucci R.A. Frank J. Nat. Struct. Biol. 1999; 6: 643-647Crossref PubMed Scopus (277) Google Scholar). It is still unclear how energy is transformed into movement, although it seems to be related to rearrangements within both EF-G and the ribosome that allow the complex to act as a molecular ratchet (15Agrawal R.K. Heagle A.B. Penczek P. Grassucci R.A. Frank J. Nat. Struct. Biol. 1999; 6: 643-647Crossref PubMed Scopus (277) Google Scholar, 16VanLoock M.S. Agrawal R.K. Gabashvill I.S. Qi L. Frank J. Harvey S.C. J. Mol. Biol. 2000; 304: 507-615Crossref PubMed Scopus (83) Google Scholar). The eukaryotic system has not been so deeply studied. However, it seems to be more sophisticated, as demonstrated by the larger number of proteins constituting the eukaryotic ribosome and by the ability to regulate EF-2 function by specific kinases (17Ryazanov A.G. Shestakova E.A. Natapov P.G. Nature. 1988; 334: 170-173Crossref PubMed Scopus (344) Google Scholar) and ADP-ribosylation (18Iglewski W.J. Fendrick J.L. ADP-ribosylating Toxins and G-proteins: Insights into Signal Transduction. American Society for Microbiology, Washington, DC1990: 511-524Google Scholar). Nevertheless, the general features of the process are assumed to be the same. In coherence with this, there is a substantial degree of homology between bacterial EF-G and eukaryotic EF-2. The more relevant differences are the greater length of EF-2 and the regulatory mechanisms depicted above. Likewise, it is remarkable that the G′-subdomain has been replaced in EF-2 by another insert termed the G“-subdomain, which is 15–30 residues longer, with a position that is displaced beyond in the sequence, and shows no homology with the prokaryotic G′-subdomain (19Aevarsson A. J. Mol. Evol. 1995; 41: 1096-1104Crossref PubMed Scopus (59) Google Scholar). On the other hand, EF-2 is a highly conserved protein within the entire eukaryotic kingdom. This fact makes especially striking the existence of EF-2 inhibitors such as sordarin, which exclusively impair fungal and not bacterial, mammalian, or plant protein synthesis machinery with the added capacity to discriminate between closely related fungal species (6Justice M.C. Hsu M.J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 20Domı́nguez J.M. Kelly V.A. Kinsman O.S. Marriott M.S.,. Gómez de las Heras F. Martı́n J.J. Antimicrob. Agents Chemother. 1998; 42: 2274-2278Crossref PubMed Google Scholar, 21Shastry M. Nielsen J. Ku T. Hsu M.J. Liberator P. Anderson J. Schmatz D. Justice M.C. Microbiology. 2001; 147: 383-390Crossref PubMed Scopus (43) Google Scholar). Identification of the residues involved in sordarin binding to EF-2 might help to explain such selectivity. For this reason, we have performed photoaffinity labeling studies of EF-2 from the pathogenic fungus Candida albicans, using a radiolabeled sordarin derivative. Elongation factor 2 and salt-washed ribosomes were isolated fromC. albicans 2005E as previously described (5Domı́nguez J.M. Martı́n J.J. Antimicrob. Agents Chemother. 1998; 42: 2279-2283Crossref PubMed Google Scholar). EF-2 concentration was determined spectrophotometrically usingE 280 = 67310m−1·cm−1 as deduced from its amino acid sequence (Swiss-Prot O13430). Ribosome concentration was also calculated spectrophotometrically assuming oneA 260 unit corresponds to 18 pmol of ribosomes (22Spedding G. Ribosomes and Protein Synthesis: A Practical Approach. IRL Press, Oxford1990: 1-29Google Scholar). [14C]GM258383 (1.8 GBq/mmol) and [3H]sordarin (180 GBq/mmol) were prepared by the Isotope Chemistry Group at GlaxoSmithKline Medicines Research Center (Stevenage, United Kingdom). Non-radiolabeled sordarin derivatives were prepared by the Medicinal Chemistry Unit at GlaxoSmithKline S.A. (Tres Cantos, Spain). Sephadex G-25 (PD-10 columns) was from Amersham Pharmacia Biotech (Uppsala, Sweden). All other chemicals were from Sigma Chemical Co. Binding of [3H]sordarin to EF-2 alone or to equimolar mixtures of EF-2 and ribosomes was studied by equilibrium dialysis using microvolumetric dialysis capsules (Cellu Sep, San Antonio, TX). The two chambers of each capsule were separated by dialysis membrane with a cutoff of 6 kDa. One chamber was filled with 150 µl of [3H]sordarin at appropriate concentration in 25 mm Hepes-KOH, pH 7.4, 85 mmpotassium acetate, 4 mm magnesium acetate, and 1.5 mmdl-dithiothreitol. The other chamber was filled with the same solution containing either 3.6 µmEF-2 or 333 nm EF-2, 333 nm ribosomes, and 33.3 µm Gpp(NH)p. Samples were incubated overnight at 30 °C under rotary shaking. Finally, duplicate 50-µl aliquots were withdrawn from each chamber, and their radioactivity was measured. Free sordarin was calculated from radioactivity values of the first capsule chamber, whereas bound sordarin was calculated by subtracting the latter from each corresponding value of the second capsule chamber. The reaction was performed in Eppendorf tubes containing 50 µl of 20 µm EF-2 in 30 mm Tris-HCl, pH 7.5, 10 mm KCl, 200 µm EDTA, 20% (v/v) glycerol, 10 mmβ-mercaptoethanol and the appropriate amount of [14C]GM258383. After preincubating for 15 min at 25 °C in the dark, samples were placed on ice and irradiated at 254 nm with an UV lamp (2000 microwatts/cm2). Further sample processing was dependent on the aim of the experiment. For quantification purposes, samples were denatured by adding 150 µl of 8 mguanidine chloride followed by 5 min of heating at 80 °C. Then, free drug was removed by gel filtration through Sephadex G-25, and the extension of covalent labeling was determined by liquid scintillation counting. For other purposes samples were processed as opportunely described. Photolabeling of EF-2 was performed as described above using 100 µm[14C]GM258383 and 10-min UV-irradiation. Gel filtration on Sephadex G-25 was then used to remove drug excess and to exchange buffer to 100 mm ammonium acetate, pH 5.0. Further reduction and alkylation with 4-vinylpyridine, trypsin digestion, and reverse-phase HPLC were performed according to established methods (23Lottspeich F. Microcharacterization of Proteins. VCH, Weinheim1994: 11-130Crossref Google Scholar). Amino acid sequences were determined with an Applied Biosystems pulse-liquid sequencer model 477A connected on-line to a reverse-phase HPLC unit for identification of the stepwise released phenylthiohydantoin-amino acids. Mass determinations were performed with a Bruker Biflex III MALDI time of flight mass spectrometer. These analysis were done by Eurosequence b.v. (Groningen, The Netherlands). GM258383, the photoactivatable aryl azide sordarin derivative used in the present work (see structure in Fig.1), inhibits protein synthesis inC. albicans cell-free systems (IC50 = 2 µm, determined as in Ref. 20Domı́nguez J.M. Kelly V.A. Kinsman O.S. Marriott M.S.,. Gómez de las Heras F. Martı́n J.J. Antimicrob. Agents Chemother. 1998; 42: 2274-2278Crossref PubMed Google Scholar). This biological activity makes GM258383 appropriate for studies intended to identify sordarin binding site. Ideally, this photoprobe was intended to label the ribosome·EF-2 complex, because this is considered the functional target of sordarin. However, the spectral properties of GM258383 (maximum absorption at 260 nm and no absorption at wavelengths longer than 300 nm) are almost identical to those of ribosomes; consequently, photoactivation of the probe is quenched by the inner filter effect of ribosomes. Indeed, all efforts to photolabel mixtures of ribosomes and EF-2 were unsuccessful (data not shown). Therefore, labeling experiments were carried out with EF-2 alone. To characterize the binding of sordarin to its target we have performed equilibrium dialysis experiments with [3H]sordarin and either EF-2 alone or equimolar mixtures of EF-2 and ribosomes in the presence of a non-hydrolyzable GTP analog, i.e. Gpp(NH)p (Fig. 2). Sordarin binding to EF-2 alone showed saturation, the Scatchard plot corresponding to that of one single class of binding sites (Fig. 2 A). Fitting the experimental points to a single hyperbola yielded K d= 1.26 µm. This indicates that sordarin binds to EF-2 in a specific manner to a defined binding site within the EF-2 molecule and hence supports performing photoaffinity labeling with EF-2 alone. This affinity, similar to that described for rat liver EF-2 and its natural substrate GTP (K d = 3.0 µm) (24Sontag B. Reboud A.M. Divita G. Di Pietro A. Guillot D. Reboud J.P. Biochemistry. 1993; 32: 1976-1980Crossref PubMed Scopus (16) Google Scholar), seems to be high enough to reduce the risk of nonspecific labeling (25King S.M. Kim H. Haley B.E. Methods Enzymol. 1991; 196: 449-466Crossref PubMed Scopus (20) Google Scholar, 26Kotzyba-Hibert F. Kapfer I. Goeldner M. Angew. Chem. Int. Ed. Engl. 1995; 34: 1296-1312Crossref Scopus (388) Google Scholar). On the other hand, the presence of ribosomes unveils two classes of binding sites with higher affinity, as deduced from the Scatchard plot (Fig. 2 B). K d values for these two sites (calculated from data fitting to a double hyperbola) were 0.7 and 41.5 nm. These values are far apart with respect to the value with EF-2 alone and might be indicative of the presence of two populations of ribosomes (pre- and post-translocated) in agreement with our previous observations (4Domı́nguez J.M. Gómez-Lorenzo M.G. Martı́n J.J. J. Biol. Chem. 1999; 274: 22423-22427Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The affinity increase may result from conformational changes within EF-2 upon interaction with the ribosome rather than from the creation of a new site in EF-2, because the latter seems unlikely in view of the already notable affinity shown by EF-2 alone. The possibility of a combined binding site in the interface between ribosome and EF-2 cannot be ruled out either. Interestingly, it has been recently shown that the sordarin derivative GM193663 increases the reactivity of 26 S rRNA inSaccharomyces cerevisiae ribosomes, most notably affecting to the sarcin-ricin loop, which becomes more exposed (27Briones C. Ballesta J.P.G. Nucleic Acids Res. 2000; 28: 4497-4505Crossref PubMed Scopus (11) Google Scholar). The use of aryl azides (such as GM258383) in photoaffinity labeling is widespread. Nevertheless, the long lifetime of the reactive intermediate has been deemed a serious caveat that may lead to nonspecific labeling (28Chowdhry V. Westheimer F.H. Annu. Rev. Biochem. 1979; 48: 293-325Crossref PubMed Scopus (706) Google Scholar). Fig.3 A shows EF-2 photolabeling with [14C]GM258383 at different irradiation times. Labeling followed pseudo-first order kinetics withk obs of 0.215 min−1 and was quantitatively completed after 10 min. When EF-2 was irradiated in the presence of increasing concentrations of [14C]GM258383, a hyperbolic saturation curve was obtained (Fig. 3 B), with maximum incorporation at 0.39 mol/mol EF-2. The most convincing result to support the specificity of photolabeling with [14C]GM258383 is presented in Fig. 3 C, where it is demonstrated that sordarin derivative GM193663 prevented photolabeling in a dose-dependent manner, so that incorporation of the photoprobe was completely precluded in the presence of high concentrations of GM193663 (Fig.4, lane 3). On the other hand, when the reaction mixture was not irradiated at 254 nm no labeling was detected by SDS-polyacrylamide gel electrophoresis and fluorography (Fig. 4, lane 4). In all, it is proved that GM258383 is covalently bound to a EF-2 sordarin binding site as a result of a photoactivatable process.Figure 4Fluorography of samples from EF-2 photolabeling. 20 µm EF-2 was incubated with 100 µm [14C]GM258383 and either not exposed to UV light (lane 4) or UV-irradiated for 10 min in the presence (lane 3) or absence (lane 2) of 10 mm GM193663. Samples were then diluted 20-fold with denaturing electrophoresis sample buffer, and 5 µl was loaded on a 10% acrylamide gel. After SDS-polyacrylamide gel electrophoresis was run the gel was dried and subjected to fluorography. Lanes 1and 5, molecular weight markers.View Large Image Figure ViewerDownload (PPT) To identify the EF-2 residue modified by [14C]GM258383, photolabeled EF-2 was digested with trypsin and the resulting peptide mixture was resolved by reverse-phase HPLC (Fig.5). Five fractions (named “A” to “E”) showed significant radioactive levels, which accounted for more than 60% of the total. Besides, there is a spurious trail of radioactivity at the end of the chromatogram, which is not associated with UV peaks at 214, 254, 280, or 297 nm; hence, it might be attributed to traces of free decomposed radioligand. Fraction A was further purified by HPLC (Fig.6) and yielded two radioactive peaks, “A1” and “A2.” Sequence analysis of these two peaks, by Edman degradation, rendered uFANxYSK (peak A1; where “u” denotes no unambiguous assignment and “x” denotes no detection) and FANxYSKK (peak A2). MALDI-MS of peak A1 rendered a signal atm/z = 1437 (MH+), which could be attributable to the mass of the peptide QFANKYSK (986 Da) plus an added mass of 450 Da corresponding exactly to the expected mass increase due to the linked photoprobe (Fig. 7). Likewise, MALDI-MS of peak A2 gave a signal atm/z = 1437 (MH+), attributable to the mass of FANKYSKK (986 Da) plus the photoprobe. Such sequences correspond to the fragment Gln224–Lys232within the C. albicans EF-2 sequence, Lys228being the modified residue. Modification of this lysine residue, which explains that trypsin did not cleave at this position and that the residue was not identified after Edman degradation, is the result of one of the mechanisms described for the photochemistry of aryl azides. As depicted in Fig. 7, it proceeds via triplet nitrene, leading to abstraction of a hydrogen radical from the protein and further combination of the newly formed radicals to yield the covalent bond. The hydrogen radical is preferably abstracted from a carbon atom adjacent to heteroatoms of the amino acid side chain, as is the case of Cε in Lys (26Kotzyba-Hibert F. Kapfer I. Goeldner M. Angew. Chem. Int. Ed. Engl. 1995; 34: 1296-1312Crossref Scopus (388) Google Scholar).Figure 6Purification of fraction A. Fraction A from the first HPLC run (Fig. 5) was subjected to re-chromatography on the same column using a linear gradient of 0–90% acetonitrile in 0.05% (w/v) ammonium acetate, pH 6.0, during 120 min at 0.35 ml/min. Fractions were collected for different intervals and one-third of their volume was used for radioactivity counting by liquid scintillation.A, UV profile of the column eluate; arrows denote positions of the peaks that were selected according to the content of radioactivity. B, radioactivity expressed as total cpm in each of the collected fractions.View Large Image Figure ViewerDownload (PPT)Figure 7Reactions involved in EF-2 photolabeling with [14C]GM258383. Reactions are inferred from the experimental results of this paper, according to the photochemistry of aryl azides described in Ref. 26Kotzyba-Hibert F. Kapfer I. Goeldner M. Angew. Chem. Int. Ed. Engl. 1995; 34: 1296-1312Crossref Scopus (388) Google Scholar.View Large Image Figure ViewerDownload (PPT) The same procedure was followed with fraction B. Further chromatography yielded a single radioactive peak (Fig.8). No sequence could be obtained from Edman degradation of this peak. However, MALDI-MS revealed one signal at m/z = 1419 (MH+) attributable to the mass of qFANKYSK (968 Da; where “q” is pyroglutamic acid) plus the mass of the photoprobe (450 Da). Transformation of Gln into Glu and subsequent lactam formation blocked the N terminus of the peptide, thus preventing Edman degradation. Finally, when the rest of the radioactive peaks were analyzed in the same form, either no radioactivity was detected in the re-chromatography, or no sequence nor MS results were obtained. This suggests that these peaks might correspond to modified peptides present in scarce amounts. The modified Lys228 is located in the G-domain (also called domain I) of EF-2, more precisely at the beginning of the insert called G“-subdomain. The analog insert in bacterial EF-G (called the G′-subdomain) is located before the G5 motif, between helix DG and strand 6G (see Ref.9). However, in eukaryotic EF-2, the insert is longer and placed after the G5 motif. Because this G”-subdomain is involved in sordarin binding, such differences between G“- and G′-subdomains can play a role in determining the innocuousness of sordarins on bacterial protein synthesis. Fig. 9 shows the alignment of the G“-subdomains from all EF-2 sequences presently available. It is noteworthy that such a subdomain is longer in EF-2 from higher animals than in the rest of eukaryotic species due to a 13-residue fragment located at the beginning of the subdomain. More remarkable is the existence of two putative Walker motifs (29Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4268) Google Scholar), with Walker-A being present only in this group of species. Recently, Gonzalo et al. (30Gonzalo P. Sontag B. Lavergne J.P. Jault J.M. Reboud J.P. Biochemistry. 2000; 39: 13558-13564Crossref PubMed Scopus (10) Google Scholar) identified in rat liver EF-2 a second nucleotide binding site specific for ATP, which may probably involve these additional Walker motifs. Although the underlying physiological function is not known, these observations show that, despite the high homology, significant differences exist in G”-subdomains among species. Thus, the high selectivity of sordarin toward one such set of species (the fungi) appears less surprising. The G“-subdomain is thought to directly interact with the sarcin-ricin loop of 26 S rRNA in fungi. The peptide identified in this work is very close to Trp218, an essential residue in the interaction with the loop (31Guillot D. Lavergne J.P. Reboud J.P. J. Biol. Chem. 1993; 268: 26082-26084Abstract Full Text PDF PubMed Google Scholar). Putting together these observations with the above-mentioned effect of GM193663 on S. cerevisiae 26 S rRNA (27Briones C. Ballesta J.P.G. Nucleic Acids Res. 2000; 28: 4497-4505Crossref PubMed Scopus (11) Google Scholar), the hypothesis of a combined binding site between ribosome and EF-2 becomes more plausible. This in turn opens the possibility that differences among ribosomes also contribute significantly to sordarin specificity. In a previous paper, a theoretical three-dimensional model for S. cerevisiae EF-2 was presented (32Capa L. Mendoza A. Lavandera J.L. Gómez de las Heras F. Garcı́a-Bustos J.F. Antimicrob. Agents Chemother. 1998; 42: 2694-2699Crossref PubMed Google Scholar). The model was built from homology mapping of EF-2 onto the crystal structure ofThermus thermophilus EF-G. In this model a sordarin binding site was proposed based on the position of the mutations conferring resistance to sordarin: 9 of 14 mutated residues (32Capa L. Mendoza A. Lavandera J.L. Gómez de las Heras F. Garcı́a-Bustos J.F. Antimicrob. Agents Chemother. 1998; 42: 2694-2699Crossref PubMed Google Scholar, 33Cook N.D. Drug Discov. Today. 2000; 1: 287-294Crossref Scopus (141) Google Scholar) mapped closely on domain III, defining a possible binding pocket. Accordingly, Shastry et al. (21Shastry M. Nielsen J. Ku T. Hsu M.J. Liberator P. Anderson J. Schmatz D. Justice M.C. Microbiology. 2001; 147: 383-390Crossref PubMed Scopus (43) Google Scholar) have recently shown in this domain a block of eight amino acids (corresponding to residues 517–524 inS. cerevisiae EF-2) that may define a sordarin specificity region in natural fungal species. We have not made use of this three-dimensional model, because the photolabeled peptide is included in the G“-subdomain, which is not present in the template EF-G molecule, and hence the accuracy of the model may not suffice for our purposes. Nevertheless, it is obvious that the putative binding region deduced from our studies is far from the pocket predicted from these resistant mutations. Recent studies strongly suggest that domain III is closely related to the G-domain both structurally and functionally. It seems to influence the GTP binding center (34Martemyanov K.A. Gudkov A.T. J. Biol. Chem. 2000; 275: 35820-35824Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and to participate in the transmission of conformational rearrangements from the G-domain to domain IV after GTP hydrolysis (35Wilson K.S. Noller H.F. Cell. 1998; 92: 131-139Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Therefore, these mutations in EF-2 may help to overcome the effect of the drug by affecting EF-2 function rather than sordarin binding to EF-2 alone, thus precluding the transition to a high affinity sordarin binding complex upon interaction with the ribosome. On the other hand, there are five more sordarin-resistant mutations out of domain III, two of them located near the photolabeled peptide (6Justice M.C. Hsu M.J. Tse B. Ku T. Balkovec J. Schmatz D. Nielsen J. J. Biol. Chem. 1998; 273: 3148-3151Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). In summary, the results presented in this paper may contribute to the sordarin mode of action. In any case, studies designed to afford a more detailed definition of the molecular effects of this drug at ribosomal level are still needed. We thank Dr. H. J. Bak and Dr. W. J. Weijer (Eurosequence b.v.) for their excellent work, and M. Nieto, R. Sarabia, and A. Monjo for their assistance." @default.
• W2042211679 created "2016-06-24" @default.
• W2042211679 creator A5000296376 @default.
• W2042211679 creator A5056626874 @default.
• W2042211679 date "2001-08-01" @default.
• W2042211679 modified "2023-10-16" @default.
• W2042211679 title "Identification of a Putative Sordarin Binding Site inCandida albicans Elongation Factor 2 by Photoaffinity Labeling" @default.
• W2042211679 cites W124504601 @default.
• W2042211679 cites W1606574816 @default.
• W2042211679 cites W1607300871 @default.
• W2042211679 cites W1810161475 @default.
• W2042211679 cites W1844993037 @default.
• W2042211679 cites W1976980348 @default.
• W2042211679 cites W1979195429 @default.
• W2042211679 cites W1979959779 @default.
• W2042211679 cites W2022072826 @default.
• W2042211679 cites W2022895983 @default.
• W2042211679 cites W2030825535 @default.
• W2042211679 cites W2031225710 @default.
• W2042211679 cites W2040982970 @default.
• W2042211679 cites W2058490786 @default.
• W2042211679 cites W2076115445 @default.
• W2042211679 cites W2083521091 @default.
• W2042211679 cites W2092633225 @default.
• W2042211679 cites W2095384140 @default.
• W2042211679 cites W2098970719 @default.
• W2042211679 cites W2105663186 @default.
• W2042211679 cites W2106882534 @default.
• W2042211679 cites W2115072457 @default.
• W2042211679 cites W2120228451 @default.
• W2042211679 cites W2128832726 @default.
• W2042211679 cites W2137351715 @default.
• W2042211679 cites W2148877718 @default.
• W2042211679 cites W2152348402 @default.
• W2042211679 cites W2177552052 @default.
• W2042211679 cites W4323053626 @default.
• W2042211679 cites W935776708 @default.
• W2042211679 cites W2155596951 @default.
• W2042211679 doi "https://doi.org/10.1074/jbc.m104183200" @default.
• W2042211679 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11402051" @default.
• W2042211679 hasPublicationYear "2001" @default.
• W2042211679 type Work @default.
• W2042211679 sameAs 2042211679 @default.
• W2042211679 citedByCount "10" @default.
• W2042211679 countsByYear W20422116792012 @default.
• W2042211679 countsByYear W20422116792022 @default.
• W2042211679 countsByYear W20422116792023 @default.
• W2042211679 crossrefType "journal-article" @default.
• W2042211679 hasAuthorship W2042211679A5000296376 @default.
• W2042211679 hasAuthorship W2042211679A5056626874 @default.
• W2042211679 hasBestOaLocation W20422116791 @default.
• W2042211679 hasConcept C104317684 @default.
• W2042211679 hasConcept C107824862 @default.
• W2042211679 hasConcept C112950240 @default.
• W2042211679 hasConcept C116834253 @default.
• W2042211679 hasConcept C139124968 @default.
• W2042211679 hasConcept C185592680 @default.
• W2042211679 hasConcept C191897082 @default.
• W2042211679 hasConcept C192562407 @default.
• W2042211679 hasConcept C2778581200 @default.
• W2042211679 hasConcept C2780917455 @default.
• W2042211679 hasConcept C33947775 @default.
• W2042211679 hasConcept C55493867 @default.
• W2042211679 hasConcept C59822182 @default.
• W2042211679 hasConcept C67705224 @default.
• W2042211679 hasConcept C86803240 @default.
• W2042211679 hasConcept C88478588 @default.
• W2042211679 hasConcept C89423630 @default.
• W2042211679 hasConceptScore W2042211679C104317684 @default.
• W2042211679 hasConceptScore W2042211679C107824862 @default.
• W2042211679 hasConceptScore W2042211679C112950240 @default.
• W2042211679 hasConceptScore W2042211679C116834253 @default.
• W2042211679 hasConceptScore W2042211679C139124968 @default.
• W2042211679 hasConceptScore W2042211679C185592680 @default.
• W2042211679 hasConceptScore W2042211679C191897082 @default.
• W2042211679 hasConceptScore W2042211679C192562407 @default.
• W2042211679 hasConceptScore W2042211679C2778581200 @default.
• W2042211679 hasConceptScore W2042211679C2780917455 @default.
• W2042211679 hasConceptScore W2042211679C33947775 @default.
• W2042211679 hasConceptScore W2042211679C55493867 @default.
• W2042211679 hasConceptScore W2042211679C59822182 @default.
• W2042211679 hasConceptScore W2042211679C67705224 @default.
• W2042211679 hasConceptScore W2042211679C86803240 @default.
• W2042211679 hasConceptScore W2042211679C88478588 @default.
• W2042211679 hasConceptScore W2042211679C89423630 @default.
• W2042211679 hasIssue "33" @default.
• W2042211679 hasLocation W20422116791 @default.
• W2042211679 hasOpenAccess W2042211679 @default.
• W2042211679 hasPrimaryLocation W20422116791 @default.
• W2042211679 hasRelatedWork W1968388290 @default.
• W2042211679 hasRelatedWork W1983196816 @default.
• W2042211679 hasRelatedWork W2139916315 @default.
• W2042211679 hasRelatedWork W2148877718 @default.
• W2042211679 hasRelatedWork W2171450081 @default.
• W2042211679 hasRelatedWork W2409891690 @default.
• W2042211679 hasRelatedWork W2427998189 @default.
• W2042211679 hasRelatedWork W4237656066 @default.
• W2042211679 hasRelatedWork W4300380013 @default.

• next