Binding of Tobramycin Leads to Conformational Changes in Yeast Trnaasp and Inhibition of Aminoacylation
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The EMBO Journal Vol. 21 No. 4 pp. 760±768, 2002 Binding of tobramycin leads to conformational changes in yeast tRNAAsp and inhibition of aminoacylation Frank Walter, Joern PuÈ tz, Richard Giege 1998). Although the global architecture of tRNAs is highly and Eric Westhof1 conserved, subtle protein±tRNA interfacial contacts guar- antee speci®c aminoacylation of each tRNA by a speci®c UPR 9002 du CNRS, Institut de Biologie MoleÂculaire et Cellulaire, synthetase. The aminoacylation reaction of yeast tRNAAsp 15 rue Rene Descartes, F-67084 Strasbourg Cedex, France (Figure 1A) by its cognate aspartyl-tRNA synthetase 1Corresponding author (AspRS) is biochemically (Romby et al., 1985; PuÈtz et al., e-mail: [email protected] 1991; Frugier et al., 1994) and structurally well described (Westhof et al., 1985; Cavarelli et al., 1993). Aminoglycosides inhibit translation in bacteria by Aminoglycosides are known to interact with ribosomal binding to the A site in the ribosome. Here, it is shown RNAs inhibiting translation at the ribosome level that, in yeast, aminoglycosides can also interfere with (Blanchard et al., 1998). They further interfere with other processes of translation in vitro. Steady-state translational control (Tok et al., 1999), viral transcription aminoacylation kinetics of unmodi®ed yeast tRNAAsp Asp of HIV (Zapp et al., 1993; Mei et al., 1995) and ribozyme transcript indicate that the complexbetween tRNA activity (reviewed in Schroeder et al., 2000). Here, the and tobramycin is a competitive inhibitor of the interaction of tobramycin, a common aminoglycoside of aspartylation reaction with an inhibition constant (KI) the 2¢deoxystreptamine group (Figure 1B), with the yeast of 36 nM. Addition of an excess of heterologous tRNAAsp±AspRS complex was investigated. It is demon- tRNAs did not reverse the charging of tRNAAsp, indi- strated that the aminoacylation of tRNAAsp, a primary step cating a speci®c inhibition of the aspartylation reac- in protein synthesis, can be speci®cally inhibited by tion. Although magnesium ions compete with the tobramycin with binding af®nities in the nanomolar range. inhibitory effect, the formation of the aspartate adeny- Tobramycin is not the ®rst compound shown to inhibit late in the ATP±PP exchange reaction by aspartyl- i aminoacylation. Purpuromycin had already been shown to tRNA synthetase in the absence of the tRNA is not inhibit the aminoacylation reaction at the level of tRNA inhibited. Ultraviolet absorbance melting experiments charging, but with several molecules of purpuromycin indicate that tobramycin interacts with and desta- binding with micromolar af®nity to all tRNAs (Kirillov bilizes the native L-shaped tertiary structure of et al., 1997). Similarly, tobramycin is not the ®rst tRNAAsp . Fluorescence anisotropy using ¯uorescein- compound shown to interfere with an RNA±protein labelled tobramycin reveals a stoichiometry of one Asp enzymic system. Recently, it has been shown that molecule bound to tRNA with a KD of 267 nM. The synthetic benzimidazole derivatives inhibit the processing results indicate that aminoglycosides are biologically of the precursor-tRNAs to mature tRNAs by the RNA effective when their binding induces a shift in a con- subunit of Escherichia coli RNase P (M1 RNA), but with formational equilibrium of the RNA. I values between 5 and 20 mM (Hori et al., 2001). Keywords: aminoacyl-tRNA synthetase/antibiotics/ 50 ¯uorescence spectrometry/pharmacology/translation Results Asp Introduction Aspartylation activity of tRNA in the presence of tobramycin Aminoacylation of transfer RNA (tRNA) is essential for Aminoacylation experiments with yeast AspRS (Figure 1) cell replication and growth. It is therefore an attractive reported here were carried out on molecules obtained target for therapeutic intervention. The following strat- in vitro by transcription with T7 RNA polymerase. Figure 2 egies have been explored so far: (i) amino acid analogues compares the aspartylation kinetics of wild-type tRNAAsp (Loft®eld, 1973; Vasquez, 1974), (ii) oligonucleotides transcript in the absence and presence of the aminoglyco- mimicking tRNA features (Loft®eld, 1973), (iii) amino- side tobramycin. The aspartylation of tRNAAsp decreases acyl-adenylate analogues (Sassanfar et al., 1996) and (iv) with increased concentrations of tobramycin (1±3 mM). blocking the formation of initiator tRNAfMet (Loft®eld, The lines in the absence and presence of the antibiotic 1973). Up to now, most of the known natural products intercept the y-axis at one point, indicating that tobramycin targeted against speci®c aminoacyl-tRNA synthetases acts as a competitive inhibitor with respect to tRNAAsp (aaRSs), or apparent amino acid speci®cities of the (Figure 2). The Michaelis±Menten parameters kcat and Km aaRSs could not be developed into an antibiotic due in the presence and the absence of tobramycin and/or total mainly to their lack of systemic bioavailability (Schimmel tRNA from yeast are summarized in Table I together with et al., 1998). The correct aminoacylation of tRNAs by the relative kinetic speci®city constants (kcat/Km)rel =(kcat/ their cognate synthetase is crucial for the accurate Km)tobramycin/(kcat/Km)±tobramycin. A more intuitive number transmission of genetic information. It is determined by is also included, namely the loss in aminoacylation speci®c structural features of the tRNAs (Giege et al., ef®ciency caused by the antibiotic. The presence of 760 ã European Molecular Biology Organization Inhibition of tRNA aminoacylation Fig. 1. Yeast tRNAAsp and the aminoglycoside tobramycin. (A) Sequence of yeast tRNAAsp transcript (Gangloff et al., 1971) showing the change of the ®rst base pair (U1±A72®G1±C72); nucleotides are numbered according to Sprinzl et al. (1998). Identity nucleotides of the aspartylation reaction are shadowed (PuÈtz et al., 1991; Frugier et al., 1994). The G1±C72 wild-type transcript shows equivalent aspartylation parameters to those of fully Asp modi®ed tRNA and U1±A72 transcripts (PuÈtz et al., 1991). (B) Structure of the aminoglycoside antibiotic tobramycin, a member of the 2¢deoxystreptamine group. The antibiotics of the aminoglycoside family result from modi®cations of neamine, a two-ring system made of 2-deoxystreptamine (called ring B or II) glycosylated at the 4-position by a 6-membered amino-sugar (called ring A or I) of the glycopyranoside series. Further modi®cations with various amino-sugars at the 6-position lead to the kanamycin family. aminoacylation kinetics (data not shown). Tobramycin does not exhibit a time dependence of the inhibition of the aminoacylation reaction of tRNAAsp. Thus, the kinetics measurements indicate that binding of tobramycin leads to spatial conformational changes of the tRNA, resulting in a reduced af®nity of tRNAAsp for AspRS or a loss in transition state stabilization of the tRNAAsp±AspRS com- plex. To determine whether the inhibition is speci®c for the tRNAAsp±AspRS interaction, the effect of tobramycin on tRNAAsp was determined in the presence of an increasing excess of competitor tRNA. For this purpose tRNAAsp is incubated together with up to 2-fold excess of tRNAPhe over tobramycin. No recovery of the aminoacylation Fig. 2. Inhibition of the aspartylation reaction of tRNAAsp transcripts activity is observed after the addition of native tRNAPhe by tobramycin. The double reciprocal plot (Lineweaver±Burk) shows (Figure 3A). In the high concentration range of competitor the initial velocity of the aspartylation reaction as a function of tRNA an inhibition effect of the aminoacylation reaction is tRNAAsp concentration in the absence of tobramycin (circles) and in the presence of tobramycin at 1 (squares), 2 (diamonds) and noticeable, probably due to the addition of high concen- 3 mM (triangles). trations of charges and salt. Moreover, assays in the presence of high levels of total tRNA (3- to 50-fold excess over tobramycin) also resulted in no recovery of tobramycin affects mainly the Km by factors up to 30-fold aspartylation activity (Table I). Further analysis of the (for 3 mM tobramycin at an ATP:Mg2+ ratio of 5:15 mM), kinetic parameters shows only small effects on the while the kcat is only decreased 2-fold. As a consequence aspartylation reaction. A loss of speci®city by a factor of the relative speci®city constants are decreased and the loss 8 is observed, but remains essentially unchanged with in aminoacylation ef®ciency increases up to 60-fold. increasing concentrations of total tRNA. Further, aminoacylation experiments using fully modi®ed The next question addressed was whether tobramycin yeast tRNAAsp reveal that tobramycin inhibits charging to can speci®cally inhibit tRNAAsp charging within a mixture the same extent, showing equivalent kinetic parameters for of various tRNA families. The level of aspartylation and aminoacylation compared with those of the corresponding phenylalanylation within a fraction of total tRNAs was in vitro transcripts (data not shown). The addition of monitored in the absence and presence of tobramycin. tobramycin at various times or directly before the start of Figure 3B reveals that tobramycin exhibits the same the reaction by AspRS does not show a change in the inhibition potential towards the aspartate system as shown 761 F.Walter et al. Table I. Kinetic parameters for aspartylation of yeast tRNAAsp transcripts with yeast AspRS in the absence or presence of tobramycin ±1 ATP/MgCl2 Inhibitor Competitor Km (nM) kcat (s ) kcat/Km Loss of (mM) tobramycin tRNAtotal (relative) speci®city (mM) [mM] (x-fold) 5/15a 0 ± 44 0.66 1 1 5/15a 1 ± 466 0.33 0.047 21 5/15a 2 ± 945 0.38 0.027 37 5/15a 3 ± 1190 0.30 0.017 60 5/15a 3 10 151 0.27 0.12 8 5/15a 3 30 115 0.21 0.12 8 5/15a 3 90 95 0.18 0.13 8 2/10b 0 ± 732 0.31 0.028 35 2/10b 3 ± 1285 0.58 0.03 33 aATP:Mg2+ ratio of 1:3. bATP:Mg2+ ratio of 1:5. phenylalanylation of tRNAPhe within the same pool is not affected (Figure 3B).