Direct Crosslinking of the Antitumor Antibiotic Sparsomycin, and Its

Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9003–9008, August 1999 Biochemistry Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes (sparsomycin-tRNA crosslink͞[125I]phenol-alanine-sparsomycin͞RNase H analysis) BO T. PORSE*, STANISLAV V. KIRILLOV*†,MARIANA J. AWAYEZ*, HARRY C. J. OTTENHEIJM‡, AND ROGER A. GARRETT*§ *RNA Regulation Centre, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83H, DK1307 Copenhagen K, Denmark; †Petersburg Nuclear Physics Institute, Russian Academy of Sciences, 188350 Gatchina, St. Petersburg, Russia; and ‡Organon International, P.O. Box 20, 5340 BH Oss, The Netherlands Communicated by Harry F. Holler, University of California, Santa Cruz, Santa Cruz, CA, May 28, 1999 (received for review March 2, 1999) ABSTRACT The antitumor antibiotic sparsomycin is a 23S-like rRNA of either free ribosomes or N-Ac-Phe-tRNA- universal and potent inhibitor of peptide bond formation and ribosome complexes (4, 5). It is also special in that it binds very selectively acts on several human tumors. It binds to the weakly, if at all, to ribosomes lacking an N-blocked aminoacyl- ribosome strongly, at an unknown site, in the presence of an tRNA (6). This effect is reciprocated in that sparsomycin N-blocked donor tRNA substrate, which it stabilizes on the stimulates PЈ-site binding of N-blocked tRNA fragments con- ribosome. Its site of action was investigated by inducing a taining the 3Ј-terminal-CCA-end (4, 7). These observations, crosslink between sparsomycin and bacterial, archaeal, and combined with the capacity of sparsomycin to inhibit protein eukaryotic ribosomes complexed with P-site-bound tRNA, on biosynthesis in the three domains of life, implies that it irradiating with low energy ultraviolet light (at 365 nm). The recognizes a universally conserved structural motif in the crosslink was localized exclusively to the universally con- peptidyl transferase center that may involve components from served nucleotide A2602 within the peptidyl transferase loop ribosomal protein and͞or rRNA. Because this motif is likely to region of 23S-like rRNA by using a combination of a primer be involved in binding of the 3Ј-terminal adenosine of the extension approach, RNase H fragment analysis, and P͞PЈ-site-bound tRNA (8), we can infer that one mechanism crosslinking with radioactive [125I]phenol-alanine-sparsomy- by which the drug acts is by stabilizing this interaction and, cin. Crosslinking of several sparsomycin derivatives, modified thereby, inhibiting movement of the 3Ј-end of the tRNA during near the sulfoxy group, implicated the modified uracil residue elongation. in the rRNA crosslink. The yield of the antibiotic crosslink was Previously, crosslinks were shown to form between sparso- weak in the presence of deacylated tRNA and strong in the mycin derivatives carrying affinity labels near the sulfoxy presence of an N-blocked P-site-bound tRNA, which, as was group (Fig. 1) and unidentified ribosomal proteins (9). Weak shown earlier, increases the accessibility of A2602 on the evidence also exists for a direct antibiotic-rRNA interaction ribosome. We infer that both A2602 and its induced confor- because haloarchaeal mutants carrying single-site mutations at mational switch are critically important both for the peptidyl positions 2438, 2451, 2499, 2500, or 2584 (lack of modification) transfer reaction and for antibiotic inhibition. This supposi- within the peptidyl transferase loop region of 23S rRNA tion is reinforced by the observation that other antibiotics that (Escherichia coli numbering) exhibit enhanced sparsomycin can prevent peptide bond formation in vitro inhibit, to differ- resistance (10–12). However, given the small size of the latter ent degrees, formation of the crosslink. effects, the mutated nucleotides are likely to produce general perturbations in the peptidyl transferase center rather than Sparsomycin (Fig. 1) is one of very few antibiotics that can constitute the sparsomycin binding site. If sparsomycin inter- inhibit protein synthesis in bacteria, archaea, and eucarya. acts with 23S-like rRNA, then mutation of its rRNA binding Moreover, the antibiotic and some of its derivatives selectively site may produce a dominant lethal phenotype that would act on several different human tumors (1). Although some side escape selection. effects can occur, including eye toxicity, active testing pro- In the present work, we investigate whether sparsomycin can grams with sparsomycin and its derivatives continue, and it has interact with rRNA or ribosomal protein. On irradiating with been used successfully as a potentiate of cis-platinum treat- low energy ultraviolet light, a direct crosslink was induced at ment of tumors (1). Sparsomycin is one of several antibiotics the universally conserved A2602 of 23S-like rRNA within that interfere with peptide bond formation, a process that is bacterial, archaeal, and eukaryotic ribosomes whereas no central to protein biosynthesis and takes place on the large crosslink was observed to ribosomal proteins. Parallel studies ribosomal subunit (reviewed in refs. 2 and 3). For many of with sparsomycin derivatives implicated the modified uracil these antibiotics, mutational evidence and͞or rRNA footprint- moiety of the drug in the rRNA interaction. A rationale is ing studies on free ribosomes using nucleotide-specific chem- provided for the requirement of the N-blocked P-site-bound icals implicate the peptidyl transferase loop region of 23S-like tRNA for strong drug binding. rRNA, from one or more domains of life, in their binding site (reviewed in ref. 2). Here, they perturb or block peptide bond formation in subtly different ways that are only partially MATERIALS AND METHODS understood. Preparation of Ribosomes and 23S rRNA. Ribosomes and Sparsomycin differs from the other peptidyl transferase ribosomal subunits were prepared from E. coli as described drugs in that it does not produce a chemical footprint on (13) except that the final ethanol precipitation step was replaced by centrifuging in a fixed-angle rotor at 100,000 ϫ g. The publication costs of this article were defrayed in part by page charge Ribosomes were prepared from Bacillus megaterium, Halobac- payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. E-mail: garrett@ PNAS is available online at www.pnas.org. mermaid.molbio.ku.dk. 9003 Downloaded by guest on October 1, 2021 9004 Biochemistry: Porse et al. Proc. Natl. Acad. Sci. USA 96 (1999) Sparsomycin Crosslinking to Ribosomes from Different Organisms. Sparsomycin was crosslinked to ribosomes from B. megaterium, H. halobium, and S. cerevisiae as follows. Com- plexes of sparsomycin (50 mM), ribosomes (150 nM), poly(U) (1 ␮g͞pmol ribosome), and N-Ac-Phe-tRNA (1.3 mol͞mol ribosome) were formed in 20 ␮l of 20 mM Tris⅐HCl (pH 7.5), 50 mM NH4Cl, and 10 mM MgCl2 (B. megaterium and S. cerevisiae) or 70 mM Hepes⅐KOH (pH 7.8), 60 mM magnesium acetate, 3.0 M KCl, and 1 mM DTT (H. halobium) for 20 min at 37°C. Samples then were irradiated at 365 nm for 15 min and were treated as described for E. coli. Domain V of 23S-like rRNA was analyzed by reverse transcription by using regularly spaced primers (see refs. 5 and 14). 125 FIG. 1. The chemical structures of sparsomycin and the derivatives Crosslinking of [ I]Phenol-Alanine-Sparsomycin. Phe- benzyl-sparsomycin, pentyl-sparsomycin, octyl-sparsomycin, phenol- nol-alanine-sparsomycin (named according to ref. 9) was alanine-sparsomycin (Pa-sparsomycin), and iodo-phenol-alanine- dissolved in 20 ␮l 50% DMSO at 7 mM and was iodinated with sparsomycin (I-Pa-sparsomycin). [125I]NaI (100 ␮Ci, Amersham Pharmacia) by using a mild procedure (6, 19). [125I]Phenol-alanine-sparsomycin (50 ␮M) terium halobium, and Saccharomyces cerevisiae as described (5, was subsequently complexed to 70S ribosomes (40 pmol) in the 14, 15). presence of poly(U) (1 ␮g͞pmol ribosome) and N-Ac- Crosslinking of Sparsomycin to 70S Ribosomes from E. coli. [14C]Phe-tRNA (1.3 mol͞mol ribosomes) in 80 ␮lof20mM Sparsomycin (200 nM to 150 ␮M) was incubated with 70S ⅐ ␮ ⅐ Tris HCl (pH 7.5), 50 mM NH4Cl, and 10 mM MgCl2 for 20 ribosomes (30 nM to 150 nM) in 20–100 l of 20 mM Tris HCl min at 37°C, and half of the sample was irradiated at 365 nm (pH 7.5), 50 mM NH4Cl, and 10 mM MgCl2 for 20 min at 37°C. as described above. After phenol extraction and ethanol In most experiments, deacylated tRNA or N-Ac-Phe-tRNA precipitation, 7.5 pmol of both the UV-irradiated and the (1.3 mol͞mol ribosomes) was added in the presence of poly(U) ␮ ͞ control sample, each supplemented with 15 pmol of purified (1 g pmol ribosome) (16). In the antibiotic interference 23S rRNA, were digested with RNase H in the presence of experiments, other peptidyl transferase drugs were added oligonucleotides EC2563 and EC2654, as described above. The either to sparsomycin complexed with ribosomes.poly(U).N- samples were subsequently coelectrophoresed with control Ac-Phe-tRNA or directly to the ribosomal complex before samples (with no RNase H treatment) in an 8% denaturing adding sparsomycin. After complex formation, samples were polyacrylamide gel, were stained with toluidine blue, and were placed in a microtiter tray, on an ice-water bath, and were subjected to autoradiography. Additional RNase H analysis, ϫ irradiated at 254, 312, or 365 nm in a Stratalinker 1800 (5 using regularly spaced oligonucleotides on 23S rRNA (Ϸ500 8 W bulbs, Stratagene) for 1–30 min. A Petri dish (Sarstedt) nucleotides apart) revealed no further radioactively labeled was sometimes used as a filter that efficiently eliminates fragments outside of the A2602 region. Ribosomal proteins Ͻ shorter wavelengths ( 300 nm). Samples were extracted with from the crosslinked samples (5 pmol) were subjected to phenol, phenol:chloroform, and chloroform and were precip- one-dimensional SDS͞PAGE.

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