On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel

Biochimie xxx (2013) 1e8

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Research paper

On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel Petros Mamos a,1, Marios G. Krokidis a,1, Athanassios Papadas a, Panagiotis Karahalios a, Agata L. Starosta b, c, Daniel N. Wilson b, c, Dimitrios L. Kalpaxis a, George P. Dinos a, * a b c

Department of Biochemistry, School of Medicine, University of Patras, 26500 Patras, Greece Gene Center and Department of Biochemistry, University of Munich, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany Center for Integrated Protein Science Munich (CiPSM), University of Munich, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 April 2013 Accepted 4 June 2013 Available online xxx

The ribosomal exit tunnel had recently become the centre of many functional and structural studies. Accumulated evidence indicates that the tunnel is not simply a passive conduit for the nascent chain, but a rather functionally important compartment where nascent peptide sequences can interact with the ribosome to signal translation to slow down or even stop. To explore further this interaction, we have synthesized short peptides attached to the amino group of a chloramphenicol (CAM) base, such that when bound to the ribosome these compounds mimic a nascent peptidyl-tRNA chain bound to the A-site of the peptidyltransferase center (PTC). Here we show that these CAM-peptides interact with the PTC of the ribosome while their effectiveness can be modulated by the sequence of the peptide, suggesting a direct interaction of the peptide with the ribosomal tunnel. Indeed, chemical footprinting in the presence of CAM-P2, one of the tested CAM-peptides, reveals protection of 23S rRNA nucleotides located deep within the tunnel, indicating a potential interaction with specific components of the ribosomal tunnel. Collectively, our findings suggest that the CAM-based peptide derivatives will be useful tools for targeting polypeptide chain mimics to the ribosomal tunnel, allowing their conformation and interaction with the ribosomal tunnel to be explored using further biochemical and structural methods. Ó 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Chloramphenicol-derivatives Peptidyl-tRNA analogs Nascent peptidyl-tRNA mimics Ribosomal tunnel

1. Introduction The ribosome is a nano-machine that synthesizes polypeptide chains from amino acid building blocks. As the nascent polypeptide chain is being synthesized it passes through a ribosomal tunnel within the large subunit and emerges at the solvent side where protein folding occurs [1]. For many years the ribosomal tunnel was thought of as a passive conduit for nascent chains, however, accumulating evidence indicates that, in some cases, the tunnel plays a more active role [2e4]. Recently, it was shown that the tunnel is a functionally important compartment where the structure of the nascent peptide is monitored and from which specific peptides can signal the ribosome to slow its rate of elongation or even completely stop translation [4,5]. A number of genes regulated by recognition of the nascent peptide have been identified in bacteria and eukaryotes: The tna operon of Escherichia coli [6], the erm

Abbreviations: CAM-peptides, oligopeptidyl chloraphenicol analogs. * Corresponding author. Tel.: þ30 2610 969125; fax: þ30 2610 969167. E-mail address: [email protected] (G.P. Dinos). 1 These authors contributed equally to this work.

cassettes responsible for macrolide antibiotic resistance [7,8], the SecMA operon encoding the secretion proteins SecM and SecA [9], and CPA1 in Saccharomyces cerevisiae [10] are among the most wellstudied cases. In all these cases, translational stalling occurs within an upstream open reading frame (uORF), which leads to regulation of expression of the downstream gene(s). Mutations within the upstream gene as well as within components of the ribosomal tunnel alleviate the translational arrest, indicating that the translational arrest results from a specific interaction between the nascent polypeptide chain and the ribosomal tunnel [4]. Moreover, the effect of specific ribosomal tunnel mutations differs for the various stalling peptides, which coupled with the diverse sequences of the stalling peptides themselves, suggests that different nascent chains induce translational stalling using distinct mechanisms [4]. Based on biochemical and structural data, a number of relays have been proposed as to how interaction between the nascent polypeptide chain located deep within the ribosomal tunnel leads to inactivation or silencing of the peptidyltransferase center (PTC) [7,8,11e14]. However, higher resolution structures will be required to provide further mechanistic insight into the stalling mechanisms.

0300-9084/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2013.06.004

Please cite this article in press as: P. Mamos, et al., On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel, Biochimie (2013), http://dx.doi.org/10.1016/j.biochi.2013.06.004

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P. Mamos et al. / Biochimie xxx (2013) 1e8

Here we provide a simple methodology to target nascent polypeptide chains to the ribosomal tunnel by chemically linking oligopeptides of diverse sequence to the antibiotic chloramphenicol (CAM, Fig. 1). Since CAM binds to the large ribosomal subunit within the A-site of the PTC (Fig. 1), this enables the attached oligopeptide sequences to be delivered to the ribosome in a manner such that they can mimic a nascent polypeptide chain extending into the ribosomal tunnel. Here we demonstrate that these CAMpeptides bind at the PTC of the ribosome by showing that they inhibit translation, the puromycin reaction, and compete with binding of radiolabelled-CAM to the ribosome. The fact that the effectiveness of the CAM-peptides is influenced by the sequence of the attached oligopeptide, suggests an interaction of the peptide moiety with the ribosomal tunnel. Consistently, we also observed chemical protection of 23S rRNA nucleotides located within the ribosomal tunnel. Collectively, these findings indicate that CAMpeptides will be useful for positioning oligopeptides of specific sequence within the ribosomal tunnel in order to mimic nascent polypeptide chains, and thereby investigate mechanisms of polypeptide-mediated translation regulation. 2. Materials and methods 2.1. Materials L-[2,3,4,5,6-3H]-Phenylalanine and L-[4,5-3H]-lysine were purchased from Amersham Pharmacia Biotech (Piscataway, NJ), whereas [14C]-ERY and [14C]-chloramphenicol ([14C]-CAM) were obtained from Perkin Elmer (USA) and Moravek Biochemicals (Brea, CA), respectively. [g-32P]ATP was purchased from Izotop (Hungary). Avian myeloblastosis virus-reverse transcriptase and T4 polynucleotide kinase were from Roche Diagnostics GmbH (Mennheim, Germany). Telithromycin was kindly supplied by Aventis-Pharma to D. L. Kalpaxis. CAM free base [D-()threo-1-(p-nitrophenyl)-2amino-1,3-propanediol], the solvents and all rest reagents were purchased from Merck and Fluka. 2-chlorotrityl chloride resin and 9-fluorenylmethoxycarbonyl protected amino acids were a gift from CBL Patras, Company. 2.2. Preparation of CAM-peptides The analogs were synthesized stepwise by Fmoc solid phase methodology utilizing a 2-chlorotrityl-chloride resin [15] as solid support (Fig. 2). After protection of the CAM free base (1) with 9fluorenylmethoxycarbonyl chloride, the product 9-fluorenylmethyloxycarbonyl-protected CAM base (2) was coupled with 2chlorotrityl-chloride resin (3) through an ether bond in the

presence of diisopropylethylamine/dichloromethane (DIPEA/DCM). The product Fmoc-CAM base-resin (4) was first treated with 20% piperidine in dimethylformamide (DMF) to remove the Fmoc group and subsequently after washing the first Fmoc-amino acid coupling followed (5). Coupling was performed in situ with DIC/HOBt/DMF (diisopropylcarbodiimide/1-hydroxybenzotriazole/dimethylformamide in a 3:3.3:4.5 M ratio, respectively) for 2.5 h at room temperature. Completeness of the reaction was monitored by the Kaiser test and thin layer chromatography on 60 Merck 60F254 films (0.2 mm) pre-coated on aluminium foil. The cycle of deprotection, washing and coupling step was repeated as many times as the number of the coupled amino acids. For amino acids with side-chain amino groups, additional distinct protection groups were employed to prevent linkage of the polypeptide through the side chain amino group. For arginine in CAM-P1, we employed N-a-Fmoc-NG(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine (Fmoc-Arg(Pbf)-OH and deprotection was achieved by incubation with a mixture of TFA/TES/DCM (trifluoroacetic acid/triethylsilane/ dichloromethane in a ratio of 85:5:10, respectively) for 3 h at RT at a concentration of 10 mg/ml [16]. After the final coupling step (7), fully protected fragments were cleaved from the resin and sidechain deprotection was accomplished to liberate the fully deprotected peptidyl analogs (8), under treatment with TFA:DCM:TES:DTT (85:5:5:5) at RT for 2 h, as described elsewhere [17]. All the crude products were further purified by semi-preparative reversephase high performance liquid chromatography (Millenium 2.1 system, Nucleosil C-18 reversed phase, column 250  10 mm with 7 mm packing material, Agilent Technologies, Waldbronn, Germany). The final verification of the pure CAM-peptides was achieved by analytical RP-HPLC (Waters system equipped with a 600E controller and a Waters 2996 photodiode array UV detector) and electrospray ionization e mass spectroscopy (Waters Micromass ZQ spectrometer, equipped with a quadropole detector, coupled to a MassLynx NT 2.3 data system). 2.3. Biochemical preparations Re-associated 70S ribosomes were prepared from E. coli K12 cells as described previously [18], and were kept in buffer containing 20 mM HEPES/KOH (pH 7.6), 50 mM CH3COONH4, 6 mM (CH3COO)2Mg, and 4 mM b-mercaptoethanol. The S100 fraction was prepared as previously described [19], and was treated with DE-52 cellulose in order to remove tRNAs and RNases. EF-G was isolated from E. coli as previously described [20]. Ac[3H]Phe-tRNA was prepared using specific tRNA under standard conditions [19] and was freed of uncharged tRNA by reverse-phase HPLC on Nucleosil column using a programmed binary gradient of solvents 1

Fig. 1. Binding site of chloramphenicol on the ribosome. (a) Chemical structures of chloramphenicol (CAM) and CAM-base (* indicates the position, where the peptide moieties are attached to generate CAM-peptides). (b) Binding site of CAM (orange) to at the PTC of the ribosome, relative to Phe-tRNA (blue, [48]) at the A-site and TnaC peptidyl-tRNA (green, [11]) at the P-site. The relative position of erythromycin (ERY, tan) is also indicated (24).399.

Please cite this article in press as: P. Mamos, et al., On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel, Biochimie (2013), http://dx.doi.org/10.1016/j.biochi.2013.06.004

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Fig. 2. Schematic for the synthesis of CAM-peptides. Refer to Experimental Procedures for details.

(20 mM ammonium acetate, pH 5.0, 10 mM magnesium acetate, 400 mM NaCl) and 2 (60% v/v methanol in buffer 1). Complex C, i.e. the [70S ribosome$MF-mRNA$Ac[3H]Phe-tRNA] complex, was prepared as described previously [20]. Defined ribosomal complexes were prepared and titrated with puromycin as previously reported [20].

2.6. Poly(A)-dependent poly(Lys) synthesis This assay was performed as described for poly(Phe) assay, except that poly(A) replaced poly(U) and [3H]lysine (3 nmols, 500 dpm/pmol) replaced phenylalanine. Incubation was performed at 37  C for 1 h. The TCA solution used for polylysine peptides precipitation was treated with 0.25% sodium wolframate before use.

2.4. In vitro coupled transcriptionetranslation assay 2.7. Puromycin reaction All coupled transcriptionetranslation experiments were performed using the E. coli lysate-based system for the expression of the green fluorescence protein (GFP) type cyc3 in the presence and absence of antibiotics, as described elsewhere [20,21]. Each reaction contained 2.4 ml E. coli lysate, 2 ml reaction mix, 2.4 ml amino acids, 0.2 ml methionine, 1.8 ml reconstitution buffer, 0.2 ml pIVEX2.2-GFPcyc3 (1 mg/ml), and 1 ml antibiotic or control buffer. After incubation at 30  C with shaking (900 rpm) for 4e5 h, 2 mL of each reaction were diluted with 50 mL of buffer containing 10 mM HEPES/KOH (pH 7.8), 10 mM MgCl2, 60 mM NH4Cl, and 4 mM ßmercaptoethanol, mixed, and then transferred into black 96-well chimney flat bottom microtiter plates. The GFP fluorescence was read on a Tecan InfiniteÒ M1000 (excitation wavelength of 395 nm and emission 509 nm) and represented graphically using SigmaPlot (Systat Software, Inc.). All measurements were repeated in triplicate and had a standard deviation of less than 10%. 2.5. Poly(U)-dependent poly(Phe) synthesis The assay was carried out in buffer A. Tight-coupled 70S ribosomes (0.5 mM) were preincubated for 10 min at 37  C with each antibiotic at the appropriate concentration, in a 15 ml mixture containing 25 mg poly(U) mRNA, [3H]phenylalanine (5 nmoles, 50 dpm/pmol), 1 A260 unit tRNA bulk (E. coli), 3 mM ATP, 1.5 mM GTP, 5 mM acetyl phosphate and an optimized amount of S-100 fraction. After an incubation of 15 min at 37  C, a hot TCA precipitation was performed and polypeptides were isolated on glass fiber filters [22]. The remaining radioactivity on the filters was measured in a liquid scintillation counter. Phe incorporation was expressed as pmols of Phe incorporated per pmol 70S ribosomes, as a function of time.

The reaction between defined ribosomal complexes and an excess of puromycin in buffer A (20 mM HEPES/KOH pH 7.6, 150 mM CH3COONH4, 4.5 mM (CH3COO)2Mg, and 4 mM b-mercaptoethanol, 2 mM spermidine and 0.05 mM spermine) was carried out at 37  C for 2 min. The reaction volume was 20 ml with a final puromycin concentration of 1 mM. The reaction was stopped by the addition of an equal volume of 0.3 M sodium acetate pH 5.5 saturated with MgSO4, extracted with 1 ml of ethyl acetate, and the radioactivity contained in 700 ml of the organic phase was quantified by liquid scintillation. 2.8. Competition of [14C]-CAM binding Re-associated 70S ribosomes (0.20 mM final concentration) were incubated in buffer A with [14C]-CAM (150 dpm/pmol) at the appropriate/indicated concentration. After incubation for 10 min at 37  C, the mixture was diluted with 3 ml cold buffer A and was filtered through a 25-mm diameter cellulose nitrate membrane filter (Millipore 0.45 mm pore size). The filter was immediately washed twice with 3 ml of cold buffer A and the bound radioactivity was determined by measuring the radioactivity bound on the filter. Next, the binding of [14C]-CAM was studied in competition with cold CAM or CAMpeptides, by maintaining a constant concentration of [14C]-CAM (0.6 mM) and increasing concentration of non-radioactive competitors. Similar methods were also followed in the case of [14C]-ERY. 2.9. Antibiotic probing and chemical modification Aliquots of 70S ribosomes, 50 pmoles per tube, were incubated with and without antibiotics at 37  C. The incubation took place in

Please cite this article in press as: P. Mamos, et al., On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel, Biochimie (2013), http://dx.doi.org/10.1016/j.biochi.2013.06.004

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Table 1 CAM-peptide sequences and dissociation constants. Name

Peptide sequencea

Ki (mM)

CAM CAM-P1 CAM-P2 CAM-P3 CAM-P4

e Met-Arg-Leu-Phe-Val-CAM (MRLFV-CAM) Met-Tyr-Phe-Phe-Val-CAM (MYFFV-CAM) Ser-Thr-Phe-Tyr-Gly-CAM (STFYG-CAM) Tyr-Thr-Ser-Phe-Gly-CAM (YTSFG-CAM)

1.7 36.0 14.4 4.2 2.5

a

Sequences red from N- to C-terminus.

buffer containing 80 mM HEPES-KOH (pH 7.8), 20 mM MgCl2,100 mM NH4Cl and 1.5 mM dithiothreitol for 10 min, followed by 10 min at 20  C. After cooling on ice, modification took place by adding 2 mL dimethyl sulfate (DMS) diluted 1:5 in ethanol and incubating for 10 min at 37  C. The DMS reactions were stopped by adding 25 ml of stop solution (1 M TriseHCl, pH 7.5, 1 M b-mercaptoethanol, 0.1 mM EDTA), followed by ethanol precipitation. The pellets were resuspended in 50 mL of buffer containing 10 mM TriseHCl (pH 7.5),100 mM NH4Cl, 5 mM EDTA and 0.5% SDS, and then extracted with equal volumes of phenol, phenolechloroform and chloroform. The ribosomal RNA was precipitated with ethanol and resuspended in water. 2.10. Primer extension The modifications in the 23S rRNA were monitored by primer extension analysis using reverse transcriptase and 50 labeled primers. The cDNA products of the primer extension reactions were separated on 6% polyacrylamide/7 M urea sequencing gels. Gels were scanned with a PhosphorImager-type Fujifilm (FLA-3000; Berthold) and analyzed with ImageQuant software AIDA (Raytest). The positions of the stops in cDNA synthesis were identified by comparison with dideoxy sequencing reactions on 23S rRNA that were run in parallel [23]. 3. Results and discussion 3.1. Synthesis of novel CAM-peptides The chemical structure of CAM comprises a para-nitrophenyl ring attached to a dichloroacetamido tail (Fig. 1a). According to

the recent crystal structures [24e26], the para-nitrophenyl ring overlaps the binding site of aminoacyl moiety of an A-site bound tRNA, with the dichloroacetoamido “tail” directed toward the ribosomal tunnel (Fig. 1b). Thus, we postulated that replacing this dichloroacetoamido “tail” with short peptides would generate novel CAM-peptides, which would still bind to the A-site of the ribosomal PTC via the para-nitrophenyl ring, thus directing the attached polypeptide chain mimic into the ribosomal tunnel. The feasibility of this approach has been previously demonstrated via inhibition of puromycin reaction by short dipeptidyl-CAM analogues, such as Phe-CAM, Gly-CAM and PheGly-CAM [27e29]. In the present study, however, we wanted to develop a chemical synthesis method that would enable longer CAM-peptides to be generated without limitation on the desired amino acid sequence and composition. To do this, we utilized the CAM base, which lacks the distal dichloroacetyl moiety present in the CAM tail (Fig. 1a), as the starting scaffold. First, the amino group of the CAM base was protected using Fmoc and then tethered to a 2-chlorotrityl-chloride solid phase support via the primary hydroxyl group (Fig. 2). Subsequent deprotection (Fmoc removal) of the Fmoc-CAM base allowed coupling (peptide bond formation) between the free amino group of the CAM base and the carboxyl of the first incoming Fmoc protected amino acid (aa1), resulting in the aminoacyl-CAM product CAM-aa1 (Fig. 2). Thus, a subsequent round of washing, deprotection and Fmoc-aa2 coupling resulted in the synthesis of CAM-dipeptides (CAM-aa1-aa2). Using this approach repeatedly with different amino acids, we were able to generate homogeneous CAM-peptides of 4e15 amino acids in length with a range of diverse sequences. It should be noted that amino acids with side chain amino groups, such as arginine, require the use of additional protection groups to avoid linkage of subsequent amino acids to sidechain rather than backbone amino group. In the case of CAM-P1 which contains arginine (Table 1), Fmoc-Arg(Pbf)-OH was used [16], requiring an additional round of deprotection with TFA/TES/ DCM (see Materials and methods). All synthesized CAM-peptides were analyzed by HPLC and mass-spectrometry (MS), which revealed them to have the expected mass, as well as being homogenous with respect to peptide length. The chain sequence and length was based on previous work of Mankin group on E and K peptides that confer resistance to erythromycin or ketolides respectively [30], and many modifications followed in order to

Fig. 3. The chemical structure and mass spectrum for Tyr-Thr-Ser-Phe-Gly-CAM (YTSFG-CAM) peptidyl chloramphenicol base compound.

Please cite this article in press as: P. Mamos, et al., On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel, Biochimie (2013), http://dx.doi.org/10.1016/j.biochi.2013.06.004

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Fig. 4. Differential inhibitory effects of CAM-peptides on in vitro translation. (a) Fluorescence produced by translation of GFP in an in vitro E. coli transcriptionetranslation assay in the absence or presence of increasing concentrations of CAM (C), CAM-P1 (:), CAM-P2 (-), CAM-P3 (;), or CAM-P4 (A). The fluorescence of GFP in the absence of antibiotic corresponds to a value of 100%. (b,c) Effect of CAM and CAM-peptides on in vitro synthesis of (b) poly(U)-dependent poly(Phe) and (c) poly(A)-dependent poly(Lys). The concentration of each antibiotic was 50 mM and the final product was expressed as pmoles of incorporated amino acid per picomole of ribosomes during the time of incubation.

select the most effective among them (Table 1). The structure and mass spectrum of such a CAM-peptide is presented in Fig. 3. 3.2. CAM-peptides inhibit in vitro translation As an initial screen for the potential of the various CAM-peptides to interact with the ribosome, we employed an in vitro E. coli lysatebased coupled transcriptionetranslation system to determine the inhibitory activity of the CAM-peptides [20,21]. Specifically, the ability of the compounds to inhibit the synthesis of green fluorescence protein (GFP) was assessed by monitoring the fluorescence of GFP in the presence of increasing concentrations of each CAMpeptide, and compared to control reactions with either the CAM base or CAM. As expected, CAM is an excellent inhibitor of translation, inhibiting GFP synthesis with a half-inhibition concentration (IC50) of w0.5 mM (Fig. 4a). In contrast, the CAM-base appears to be a poor inhibitor (data not shown), producing only <10% inhibition, even at high concentration (100 mM), in agreement with previous studies [28]. This is also consistent with the recent crystal structures of CAM on the E. coli ribosome, where the dichloroacetyl tail makes an hydrogen-bond interaction with A2062 of 23S rRNA [24]. Similarly, none of the peptides CAM scaffold was efficient in GFP synthesis, nor in any other test which, will be discussed below. In comparison, we screened a number of CAM-peptides ranging in size from 4 to 10 amino acids and identified four distinct CAMpentamers (CAM-P1 to CAM-P4; Table 1), where the addition of oligopeptides to the CAM base increased its effectiveness, although none of the CAM-peptides were as effective as CAM (Fig. 4a). Interestingly, the sequence of the oligopeptide appeared to influence the inhibitory activity of the CAM-peptide, since the extent and shape of the inhibitory curves was quite distinct for each of the tested CAM-peptides (Fig. 4a). To test this further, we assessed the ability of CAM, CAM-base and the four CAM-peptides to inhibit the in vitro synthesis of poly(U)-dependent poly(Phe) as well as poly(A)-dependent poly(Lys) (Fig. 4b and c). In the absence of any compound, the formation of 600 Phe/ribosome (Fig. 4b) and 60 Lys/ ribosome (Fig. 4c) was observed in the respective translation systems. The presence of 50 mM CAM had only a minor inhibitory affect on poly(Phe) synthesis (w20%), whereas a more prominent inhibitory affect was seen on poly(Lys) synthesis (w50%). This is in agreement with previous findings [31e33] that led to the suggestion that tRNAs bearing large aromatic amino acids, such as Phe, can displace CAM from the ribosome, whereas tRNAs with smaller or charged amino acids, such as Gly or Arg, are less effective in displacing CAM (see ref. 34). Surprisingly, we observed that at the same final concentration (50 mM), some CAM-peptides were more effective inhibitors than CAM (Fig. 4b and c). For example, unlike

CAM, CAM-P3 and CAM-P4 exhibit a strong inhibitory affect on poly(Phe), reducing the Phe incorporation by 50% and 70% respectively (Fig. 4b). Similarly, CAM-P2, -P3 and -P4 reduced poly(Lys) synthesis by 65e80%, whereas the same concentration of

Fig. 5. Competition of CAM-peptides with puromycin and CAM. (a) AcPhe-Puromycin formation was measured in the absence (control) and presence of 15 mM of each antibiotic. Ribosomal complex C carrying Ac[3H]Phe-tRNA at the P-site of MF-mRNA programmed 70S ribosomes was reacted with 1 mM puromycin at 37  C for 2 min. (b) Competition of [14C]-CAM binding to vacant 70S ribosomes by non-radioactive CAM or CAM-peptides. Data were presented as dpms of bound CAM per 10 pmol of ribosomes, versus the concentration of CAM (C), CAM-P1 (:), CAM-P2 (-), CAM-P3 (;), and CAM-P4 (A).

Please cite this article in press as: P. Mamos, et al., On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel, Biochimie (2013), http://dx.doi.org/10.1016/j.biochi.2013.06.004

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CAM reduced poly(Lys) synthesis by only 50% (Fig. 4c). Collectively, these findings suggest that interaction of the polypeptide moiety of the CAM-peptides with the ribosome contributes to the binding affinity of these compounds and that the sequence of CAMpeptides plays an important role in this interaction. 3.3. CAM-peptides bind at the A-site of the PTC Next we wanted to verify that the CAM-peptides inhibit translation by binding to the A-site of the PTC, analogously to CAM (Fig. 1b). To do this, we first employed the puromycin reaction, which monitors the transfer of acetylated Phe (AcPhe) from a P-site bound AcPhe-tRNA to puromycin bound at the A-site of the PTC. As expected, the addition of CAM (15 mM) almost completely abolished this reaction (w80% inhibition; Fig. 5a), consistent with the overlap in binding position of CAM and puromycin at the PTC [34]. In this assay, 15 mM of CAM-P1 and CAM-P2 had only a minor inhibitory influence on the puromycin reaction (<10%), whereas CAM-P3 and CAM-P4 were more effective inhibitors reducing AcPhe-puromycin formation by 60% and 30%, respectively (Fig. 5a). It should also be noted that all CAM-peptides were more effective inhibitors than the CAM base, which had no effect on AcPhe-puromycin synthesis, even at high concentrations (data not shown). At higher (40 mM) concentrations, P1 and P2 were also observed to inhibit the puromycin reaction. To better ascertain whether the CAM-peptides do indeed bind analogously to CAM at the PTC of the ribosome, we performed

competition assays where [14C]-labeled CAM was pre-bound to 70S ribosomes and then displaced by the addition of increasing concentrations of non-radioactive CAM, CAM-peptides (Fig. 5b) or CAM base. Consistent with the other biochemical experiments, the CAM base did not compete at all with the [14C]-labeled CAM (data not shown), whereas the CAM-peptides could displace the radioactive CAM, with a hierarchy of efficiency such that P4 > P3 > P2 > P1. From the competition data, we were able to calculate the dissociation constant for each derivative (Table 1). In agreement with previous reports [35,36], CAM had a Kd of 1.7 mM, whereas CAM-P1 to P4 had Ki values of 36 mM, 14.4 mM, 4.2 mM and 2.5 mM, respectively. This order in affinities explains why P1 and P2 need much higher concentration compared to P1 and P2 to inhibit the puromycin reaction. The possibility of an allosteric competition by the peptide moiety seems unlikely, since the competition takes place with all peptide sequences tested and the data fit perfectly to a simple saturation curve. Moreover, each peptide lacking the CAM moiety had no effect either in competition or inhibition assays. CAM per se is a very strong inhibitor of PTase, but very poor inhibitor of poly(Phe) synthesis. In contrast, the CAM base is almost totally inactive, both in the [14C[-CAM binding and the biosynthesis assays [28,34]. Previous studies have indicated that dipeptides attached to the CAM base, such as PhePhe-CAM or Gly-Phe-CAM, are moderate inhibitors of PTase, with Ki ranging from 46 mM to 90 mM [29,37]. Our CAM-peptides also showed idiosyncratic behavior, depending on their own sequence. Nevertheless, all of them were tighter ligands of the

Fig. 6. Interaction of CAM-peptides with the ribosomal tunnel. (a) Competition of [14C]-ERY binding to vacant 70S ribosomes by non-radioactive ERY, CAM or CAM-peptides. (b) Model for the binding position of CAM-P2 with an extended conformation that establishes interaction with the A752-U2609 basepair, but not A2058/A2059. (c,d) Protection of from DMS modification of (c) domain V (nucleotides A2058 and A2059) and (d) domain II (nucleotides A751 and A752) of the 23S rRNA in the presence of indicated antibiotics. 23S rRNA was incubated for 10 min at 37  C without or with 50 mM of each antibiotic and then, modified by DMS. The lanes are numbered as following: (c) Lanes 1e4 sequence; lane 5, untreated 23S rRNA; lane 6, 23S rRNA modified by DMS; lanes 7-12, modified with DMS in the presence of: erythromycin (ERY), tylosin (TYL), chloramphenicol (CAM), chloramphenicol base (CAM-base) and CAM-P1 and CAM-P2 respectively. (d) Lanes 1e4 sequence; lane 5, untreated 23S rRNA; lane 6, 23S rRNA modified by DMS; lanes 3e8, modified with DMS in the presence of: ERY, telithromycin (TEL), TYL, CAM, CAM- base and CAM-P2, respectively.

Please cite this article in press as: P. Mamos, et al., On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel, Biochimie (2013), http://dx.doi.org/10.1016/j.biochi.2013.06.004

P. Mamos et al. / Biochimie xxx (2013) 1e8

ribosome than any dipeptidyl-CAM analog, regardless of their sequence. Compared to P3 and P4, P1 and P2 showed lower affinity for the ribosome and lower inhibitory effect in all except for GFP synthesis. The latter assay is the most natural one, since it simulates cellular conditions with all functions of transcription and translation in operation. Under such conditions, CAMpeptides may exhibit pleiotropic activities, including inhibition effects in additional steps of the translation pathway [38]. Based on this hypothesis, we could speculate that additional interactions might take place in the case of P1 and P2, occurring also in the entrance of the tunnel, where the peptide moiety reaches and causes additional clashes. Such pleotropic inhibitory effects are expected to be more pronounced in poly(Lys) and GFP synthesis, whose nascent peptide enters the ribosome tunnel. In contrast, poly(Phe) can accumulated between the subunits [39]. Inhibition is expected for AcPhe-puromycin formation. 3.4. Interaction of CAM-peptides within the ribosomal tunnel To explore the possibility that the peptide moieties of the CAMpeptides extend into the ribosomal tunnel, we performed competition assays between radioactive erythromycin (ERY) and each of the CAM-peptides (Fig. 6a). ERY is a macrolide antibiotic that binds within the upper region of the ribosomal exit tunnel, adjacent to the PTC (Fig. 1b) [24,25,40]. As expected, increasing concentrations of non-radiolabelled ERY led to dissociation of the radiolabelled ERY, producing a very low Kd of w0.1 nM, as reported previously [41e44]. As expected from the lack of any overlap between the CAM and ERY binding sites (Fig. 1b), CAM did not chase the radiolabelled ERY from the ribosome (Fig. 6a); a mild deviation from linearity was found only at very high CAM concentrations (data not shown). Previous data, supporting a competition between ERY and CAM [45], were not confirmed in our hands (Fig. 6a). Our data are more consistent with recent crystallographic data, according to which both antibiotics do not share any common area (Fig. 1, Ref. [24]), and with a proposed hypothesis supporting the idea of an allosteric interaction of CAM with the entrance of the exit tunnel [37]. Noteworthy, the affinity of ERY compared to CAM is about 2e3 order of magnitude higher. Therefore, extremely high concentrations are needed to see any competition. Similar experiments performed using the CAM-peptides also did not result in loss of ERY binding (Fig. 6a), suggesting that either the peptide moieties of the CAM-peptides do not overlap with the binding site of ERY or that the high affinity of ERY displaces the peptide moiety of the CAMpeptides. It is known that ribosomes can synthesize polypeptide chains of between 8 and 11 amino acids in the presence of ERY before translation stalls and peptidyl-tRNA drop-off occurs [46], suggesting that the five amino acids present on the CAM-peptides could still be accommodated simultaneously with ERY within the upper region of the ribosomal tunnel. In fact, recent results have shown that some specific polypeptide sequences can be completely passed through the tunnel in the presence of ERY, indicating that it is possible for both the macrolide and a polypeptide chain to cohabit the ribosomal tunnel [14]. We therefore took an alternative approach to investigate the potential interaction between the peptidyl moiety of CAM-peptides and the nascent chain exit tunnel by employing chemical footprinting. This involved monitoring the specific changes in reactivity of nucleotides within the 23S rRNA to chemical modification by dimethyl sulphate (DMS) when CAM, CAM base or CAM-P2 are present (Fig. 6c and d). The two major regions of the 23S rRNA that comprise the ribosomal tunnel were scanned for changes in reactivity: The first region encompassed nucleotides of domain V that are directly adjacent to the PTC, such as nucleotides A2058 and A2059 that comprise the ERY binding site. The second region encompassed 23S rRNA nucleotides of

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domain II that are located deeper within the ribosomal tunnel, but could still potentially interact with the peptidyl moiety of the CAMpeptide, if an extended conformation is indeed adopted. As controls, we also performed chemical modification experiments in the presence of the macrolide antibiotics ERY, telithromycin (TEL) and tylosin (TYL) (Fig. 6c and d). As expected from previous reports [41,47e49], all the macrolides tested protected A2058 and A2059 from chemical modification by DMS, whereas the presence of CAM or the CAM base did not result in any protection of these nucleotides (Fig. 6c). Also, most of the CAM-peptides did not protect A2058 or A2059, consistently with the lack of competition of these compounds with ERY (Fig. 6a). In contrast, CAM-P2 protected nucleotide A752 from DMS modification, at similar levels to those obtained by macrolides TEL and TYL (Fig. 6d), which are known to interact with [50e52] and protect these nucleotides from modification [41,52]. Specifically, in E. coli the heterocyclic side chain of TEL stacks on the U2609-A752 basepair, whereas the C14 bound mycinose in TYL interacts with this region [24,49]. In agreement with previous studies [47], the presence of ERY led to an enhancement in the reactivity of A752 (Fig. 6d). These findings lead us to suppose that, at least, the CAM-P2 peptide is oriented to the PTC in such a way that the peptide moiety extends into the ribosomal tunnel and probably establishes a defined interaction with the region surrounding nucleotide A752. It would also be possible that the protection may be indirect via interaction with U2609, which basepairs with A752, as it is presented in our model in Fig. 6b. We should note that a single substitution of Tyr in CAM-P2 to Arg abolishes any protection of nucleotide A752, although this CAM-peptide still competes effectively with [14C]-CAM for ribosome binding, suggesting that the Tyr plays an important role in establishing interactions with this region of the ribosome. 4. Conclusions Here we present a simple synthesis scheme for the generation of oligopeptidyl chloramphenicol analogs, or so called CAM-peptides. The synthesis scheme is very flexible allowing a diverse range of sequence compositions and lengths to be linked covalently to the antibiotic CAM. According to our data, the competition of [14C]CAM binding with CAM-peptides supports the notion that all compounds occupy or overlap the known binding site of CAM. Namely, the pnitro-phenyl moiety of the CAM-peptides occupies the CAM binding site at the PTC of the ribosome, orienting the attached polypeptide chain mimics to the ribosomal exit tunnel. Chemical footprinting studies did not identify clear protections in residues of the exit tunnel for most of CAM-peptides, suggesting that the peptide moiety of these compounds interacts with the tunnel in a manner that is not able to be detected by this approach, and/or that the peptides do not adopt a defined conformation within the tunnel. In contrast, we observed a clear protection from DMS modification of nucleotide A752 in domain II of the 23S rRNA in the presence of CAM-P2, suggesting that this peptide sequence (MYFFV) adopts an extended conformation reaching deep into the ribosomal tunnel and establishing a distinct interaction with this region. Similar interactions of a series of stalling nascent polypeptide chains with this region of the ribosome, such as SecM and TnaC, has been proposed to be important for their regulatory mechanism [4,6,9]. Therefore, we believe that at least some CAM-peptides could be useful tools for probing nascent polypeptide chain interaction with the ribosome, both biochemically, but also structurally. Acknowledgments We thank Professor Kleomenis Barlos CEO of CBL Patras, Company, for providing us with 2-chlorotrityl chloride resin and

Please cite this article in press as: P. Mamos, et al., On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel, Biochimie (2013), http://dx.doi.org/10.1016/j.biochi.2013.06.004

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P. Mamos et al. / Biochimie xxx (2013) 1e8

protected amino acids, Professor Dimitrios Gatos for scientific advice on peptide synthesis and Dr Sean Connell for comments on the manuscript. This work was supported by the Research Committee of Patras University, program “K. Karatheodori” (to G.D.), the Deutsche Forschungsgemeinschaft (FOR1805/WI3285/1-2), and the EMBO young investigator program (to D.N.W.). References [1] L.D. Cabrita, C.M. Dobson, J. Christodoulou, Protein folding on the ribosome, Curr. Opin. Struct. Biol. 20 (2010) 33e45. [2] C. Deutsch, The birth of a channel, Neuron 40 (2003) 265e276. [3] K. Ito, S. Chiba, K. Pogliano, Divergent stalling sequences sense and control cellular physiology, Biochem. Biophys. Res. Commun. 393 (2010) 1e5. [4] D.N. Wilson, R. Beckmann, The ribosomal tunnel as a functional environment for nascent polypeptide folding and translational stalling, Curr. Opin. Struct. Biol. 21 (2011) 274e282. [5] L.R. Cruz-Vera, M.S. Sachs, C.L. Squires, C. Yanofsky, Nascent polypeptide sequences that influence ribosome function, Curr. Opin. Microbiol. 14 (2011) 160e166. [6] F. Gong, C. Yanofsky, Instruction of translating ribosome by nascent peptide, Science 297 (2002) 1864e1867. [7] N. Vazquez-Laslop, N. Thum, A.S. Mankin, Molecular mechanism of drugdependent ribosome stalling, Mol. Cell 30 (2008) 190e202. [8] H. Ramu, N. Vázquez-Laslop, D. Klepacki, Q. Dai, J. Piccirilli, R. Micura, A.S. Mankin, Nascent peptide in the ribosome exit tunnel affects functional properties of the A-site of the peptidyl transferase center, Mol. Cell 47 (2011) 321e330. [9] H. Nakatogawa, K. Ito, The ribosomal exit tunnel functions as a discriminating gate, Cell 108 (2002) 629e636. [10] P. Fang, C.C. Spewak, C. Wu, M.S. Sach, A nascent polypeptide domain that can regulate translation elongation, Proc. Natl. Acad. Sci. U.S.A. 101 (2004) 4059e 4064. [11] B. Seidelt, C.A. Innis, D.N. Wilson, M. Gartmann, J.P. Armache, E. Villa, L.G. Trabuco, T. Becker, T. Mielke, K. Schulten, T.A. Steitz, R. Beckmann, Structural insight into nascent polypeptide chain-mediated translational stalling, Science 326 (2009) 1412e1415. [12] S. Bhushan, H. Meyer, A. Starosta, T. Becker, T. Mielke, O. Berninghausen, M. Sattler, D.N. Wilson, R. Beckmann, Structural basis for translational stalling by human cytomegalovirus (hCMV) and fungal arginine attenuator peptide (AAP), Mol. Cell 40 (2010) 138e146. [13] S. Bhushan, T. Hoffman, B. Seidelt, J. Frauenfeld, T. Mielke, O. Berninghausen, D.N. Wilson, R. Beckmann, SecM-stalled ribosomes adopt an altered geometry at the peptidyltransferase center, PLoS Biol. 9 (2011) e1000581. [14] K. Kannan, N. Vasquez-Laslop, A.S. Mankin, Selective Protein Synthesis by Ribosomes with a drug-obstructed exit tunnel, Cell 151 (2012) 508e520. [15] K. Barlos, O. Chatzi, D. Gatos, G. Stavropoulos, 2-Chlorotrityl chloride resin: studies on anchoring of Fmoc-amino acids and peptide cleavage, Int. J. Pept. Protein Res. 37 (1991) 513e520. [16] Z. Vasileiou, K.K. Barlos, D. Gatos, K. Adermann, C. Deraison, K. Barlos, Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation, Biopolymers 94 (2010) 339e349. [17] E. Krambovitis, G. Hatzidakis, K. Barlos, Preparation of MUC-1 oligomers using an improved convergent solid-phase peptide synthesis, J. Biol. Chem. 18 (1998) 10874e10879. [18] G. Blaha, U. Stelzl, C.M. Spahn, R.K. Agrawal, J. Frank, K.H. Nierhaus, Preparation of functional ribosomal complexes and the effect of buffer conditions on tRNA positions observed by cryoelectron microscopy, Methods Enzymol. 317 (2000) 292e309. [19] H.J. Rheinberger, U. Geigenmuller, M. Wedde, K.H. Nierhaus, Parameters for the preparation of Escherichia coli ribosomes and ribosomal subunits active in tRNA binding, Methods Enzymol. 164 (1988) 658e670. [20] G. Dinos, D.N. Wilson, Y. Teraoka, W. Szaflarski, P. Fucini, D. Kalpaxis, K.H. Nierhaus, Dissecting the ribosomal inhibition mechanisms of edeine and pactamycin: the universally conserved residues G693 and C795 regulate Psite tRNA binding, Mol. Cell 13 (2004) 113e124. [21] A.L. Starosta, V. Karpenko, A.V. Shishkina, A. Mikolajka, N.V. Sumbatyan, F. Schluenzen, G.A. Korshunova, A.A. Bogdanov, D.N. Wilson, Interplay between the ribosomal tunnel, nascent chain, and macrolides influences drug inhibition, Chem. Biol. 17 (2010) 504e514. [22] U. Bommer, N. Burkhardt, R. Junemann, C.M. Spahn, F.J. Triana-Alonso, K.H. Nierhaus, Ribosomes and polysomes, in: J. Graham, D. Rickwoods (Eds.), Subcellular Fractionation e a Practical Approach, IRL Press, Oxford, 1996, pp. 271e301. [23] S. Stern, D. Moazed, H. Noller, Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension, Methods Enzymol. 164 (1998) 481e489.

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Please cite this article in press as: P. Mamos, et al., On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel, Biochimie (2013), http://dx.doi.org/10.1016/j.biochi.2013.06.004