Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action Jack A. Dunklea, Liqun Xiongb, Alexander S. Mankinb, and Jamie H. D. Catea,c,1 aDepartments of Molecular and Cell Biology and Chemistry, University of California, Berkeley, CA 94720; bCenter for Pharmaceutical Biotechnology, University of Illinois, Chicago, IL 60607; and cPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Edited by Peter Moore, Yale University, New Haven, CT, and approved July 22, 2010 (received for review June 18, 2010) Differences between the structures of bacterial, archaeal, and or ketolides, yielding the so called MLSBK phenotype. Mutation eukaryotic ribosomes account for the selective action of antibiotics. of nucleotide 2057 can provide resistance to the MLSBK antibio- Even minor variations in the structure of ribosomes of different tics plus chloramphenicol (Fig. 1B) (8, 9). Other mutations (at bacterial species may lead to idiosyncratic, species-specific interac- positions 2452, 752, and 2611) described in several species can tions of the drugs with their targets. Although crystallographic confer resistance to varying subsets of these compounds (Fig. 1B) structures of antibiotics bound to the peptidyl transferase center (9, 10). or the exit tunnel of archaeal (Haloarcula marismortui) and bacter- Structural studies have greatly advanced our understanding of ial (Deinococcus radiodurans) large ribosomal subunits have been the inhibitory mechanisms of antibiotics that bind in the PTC or reported, it remains unclear whether the interactions of antibiotics exit tunnel. However, uncertainty concerning the interactions of with these ribosomes accurately reflect those with the ribosomes these compounds with rRNA and resistance phenotypes persist, of pathogenic bacteria. Here we report X-ray crystal structures of due to many significant disagreements between the reports for the Escherichia coli ribosome in complexes with clinically important antibiotics bound to either the Deinococcus radiodurans or antibiotics of four major classes, including the macrolide erythro- Haloarcula marismortui 50S ribosomal subunits (11). These dif- mycin, the ketolide telithromycin, the lincosamide clindamycin, ferences include the conformation of the macrolide ring, the con- and a phenicol, chloramphenicol, at resolutions of ∼3.3Å–3.4 Å. formation of the alkyl-aryl arm of telithromycin, the orientation Binding modes of three of these antibiotics show important varia- of the pyrrolidinyl moiety of clindamycin and the two nonover- tions compared to the previously determined structures. Biochem- lapping binding sites observed for chloramphenicol (6, 12–15). ical and structural evidence also indicates that interactions of Notably, neither H. marismortui nor D. radiodurans are closely telithromycin with the E. coli ribosome more closely resembles related to pathogenic bacteria. drug binding to ribosomes of bacterial pathogens. The present To address the differences that persist between the H. maris- data further argue that the identity of nucleotides 752, 2609, mortui and D. radiodurans structural data, we solved structures of and 2055 of 23S ribosomal RNA explain in part the spectrum four antibiotics bound to the Escherichia coli ribosome: erythromy- and selectivity of antibiotic action. cin, telithromycin, clindamycin and chloramphenicol, at resolu- tions of ∼3.3 Å–3.4 Å(Table S1). Because the E. coli ribosome has rRNA sequences similar to bacterial species of medical inter- ribosome structure ∣ erythromycin ∣ telithromycin ∣ clindamycin ∣ est, these data give a more accurate picture of the interactions be- chloramphenicol tween antibiotics and the large ribosomal subunit of pathogenic bacteria. Together with biochemical data probing the interactions ntibiotics are small organic molecules synthesized by fungi of antibiotics with the PTC and exit tunnel, the present structures Aand bacteria that can inhibit the growth of other microorgan- reveal how rRNA sequence differences contribute to the spectrum isms (1). The ribosome is a major target of antibiotics, which of activity for antibiotics and offer new clues as to why these com- affect nearly all steps of protein synthesis (2). The peptidyl trans- pounds do not inhibit cytoplasmic eukaryotic ribosomes. ferase center (PTC) of the ribosome is inhibited by a chemically diverse group of compounds including lincosamides and pheni- Results cols (Fig. 1 A–C) (3). Despite the chemical dissimilarities of these The Structure of Erythromycin Bound to the E. coli Ribosome. The ori- compounds they share overlapping mechanisms of inhibition by ginal macrolide antibiotic in clinical use since the 1950s, erythro- preventing proper orientation of tRNA in the PTC and interfer- mycin is composed of a 14-membered macrolactone ring, with ing with peptide bond formation. Another important group of carbohydrates at positions 3 and 5 (Fig. 1A). The desosamine drugs, macrolides and their modern ketolide derivatives, bind sugar at position 5, which contains a dimethyl amine that is in the exit tunnel of the large ribosomal subunit and inhibit ex- crucial for binding to the ribosome, makes contact with A2058 trusion of the nascent peptide, leading to peptidyl tRNA drop-off (Fig. 1 B and C) (4–6). Bacterial pathogens have become resistant to antibiotics that Author contributions: J.A.D., A.S.M., and J.H.D.C. designed research; J.A.D. and L.X. inhibit protein synthesis over decades of clinical use of these performed research; J.A.D., L.X., A.S.M., and J.H.D.C. analyzed data; and J.A.D., A.S.M., and J.H.D.C. wrote the paper. compounds. One of the major mechanisms of resistance is based on altering ribosomal RNA (rRNA) in the drug binding site. For The authors declare no conflict of interest. example, resistance to the macrolide erythromycin is often This article is a PNAS Direct Submission. mediated by mutations of nucleotide A2058 in 23S rRNA located Data deposition: The coordinates for the structural models have been deposited in the Protein Data Bank, www.pdb.org [PDB ID codes 3OFO, 3OFP, 3OFR, 3OFQ (70S ribosome in the site of drug action or by chemical modification of this nu- in complex with erythromycin), 3OAQ, 3OAR, 3OAS, 3OAT (70S ribosome in complex with cleotide by Erm methyltransferase, a gene that is often acquired telithromycin), 3OFX, 3OFY, 3OFZ, 3OG0 (70S ribosome in complex with clindamycin), and by bacterial pathogens (Fig. 1B) (7, 8). Remarkably, similar 3OFA, 3OFB, 3OFC, 3OFD (70S ribosome in complex with chloramphenicol)]. mutations often provide resistance to multiple protein synthesis See Commentary on page 17065. inhibitors that bind to the overlapping sites in ribosome. For 1To whom correspondence should be addressed. E-mail: [email protected]. example, mutations of 23S rRNA nucleotides 2058 or 2059 can This article contains supporting information online at www.pnas.org/lookup/suppl/ provide resistance to macrolides, lincosamides, streptogramin B, doi:10.1073/pnas.1007988107/-/DCSupplemental. 17152–17157 ∣ PNAS ∣ October 5, 2010 ∣ vol. 107 ∣ no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1007988107 in 23S rRNA, the most commonly mutated nucleotide in resistant bacteria. The 14-atom macrolactone ring of erythromycin serves SEE COMMENTARY as the scaffold for several semisynthetic compounds in clinical use, with various appendages attached to the ring. The macrolac- tone ring was initially reported in two different conformations, “folded-in” and “folded-out” when bound to D. radiodurans and H. marismortui 50S subunits, respectively (12). However, the existence of the folded-in conformation for erythromycin when bound to the ribosome has been questioned because a pu- tatively lower energy folded-out conformation of erythromycin exists in the crystal structure of the free compound (6). In the structure of erythromycin bound to the E. coli 70S ribo- some, we observed difference electron density for the drug in ex- cellent agreement with its position bound to the H. marismortui 50S ribosomal subunit containing a G2058A mutation (6) and with its conformation in the crystal structure of the free com- pound (Fig. S1) (16). A slight movement of the antibiotic relative to its position when bound to the G2058A mutant H. marismortui 50S subunit is visible, possibly due to a movement of rRNA helix H73 and the adjacent nucleotides in the E. coli ribosome, relative to their position in the H. marismortui 50S subunit (Fig. S1B). In spite of this spatial translocation, the drug maintains its contacts with A2058, which involves a hydrogen bond between the deso- samine hydroxyl and the N1 atom of A2058, and tight packing of the hydrophobic face of the lactone ring against nucleotides 2611 and 2057 in the peptide exit tunnel wall. Notably, H. marismortui contains a G-C base pair C2057-G2611 in the opposite polarity of the E. coli base pair G2057-C2611, but this seems not to affect the BIOPHYSICS AND interaction with erythromycin significantly (Fig. 1C and Fig. S1B). COMPUTATIONAL BIOLOGY A Key Moiety of Telithromycin Forms Species-Specific Contacts to rRNA. Telithromycin belongs to the family of ketolide antibiotics that represent the newest generation of macrolides. In telithro- mycin, a carbonyl group replaces the C3 cladinose sugar (Fig. 1A), which in macrolides is necessary for ribosome stalling and regu- lating the induction of resistance genes (17). Similar to other clinically relevant ketolides, telithromycin contains an alkyl-aryl