Structures of the Escherichia Coli Ribosome with Antibiotics Bound Near the Peptidyl Transferase Center Explain Spectra of Drug Action

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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–3.4Å. 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 resolutions of ∼3.3 Å–3.4 Å(Table S1). Because the E. coli ribosome ribosome structure ∣ erythromycin ∣ telithromycin ∣ clindamycin ∣ has rRNA sequences similar to bacterial species of medical interest, 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 Downloaded by guest on September 24, 2021 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 macrolactone 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 ribosome, 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 compound (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 desosamine 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 telithromycin, 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 arm attached to a carbamate heterocycle that involves the C11 and C12 positions of the ketolide macrocycle. This moiety increases the affinity of the ketolide scaffold for the ribosome by several hundred-fold, demonstrating that it is an important phar- macophore (18). The alkyl-aryl arm of telithromycin was seen in two distinct conformations in prior ribosome crystal structures. It was folded back over the macrolactone ring when bound to the G2058A H. marismortui 50S subunit, or interacting with rRNA further down the peptide exit tunnel when bound to the D. radiodurans 50S subunit (Fig. 2) (6, 13). Importantly, neither structure can easily explain telithromycin resistance mutants at residue U2609, telithromycin protection of A752 in RNA footprinting

Fig. 1. Binding sites for antibiotics in the PTC and peptide exit tunnel. (A) The chemical structures of erythromycin (a macrolide), telithromycin (a ketolide), chloramphenicol (a phenyl propanoside), and clindamycin (a lincosamide) are shown. (B) An overview of the antibiotic binding sites within the 50S subunit. Erythromycin (green), telithromycin (pink), clindamcyin (purple), and chloramphenicol (orange) are shown as stick models. Ribbons denote the sugar phosphate backbone of 23S rRNA (gray) with nucleotides of interest colored light blue, the acceptor ends of A-site tRNA (yellow) and P-site tRNA (red). The location of the peptide exit tunnel is labeled “exit,” and an icon indicating the point of view is shown on the right. (C) The secondary structure of the 3′ region of 23S rRNA showing elements of the PTC and the Fig. 2. Telithromycin bound to the E. coli ribosome. A comparison of the adjacent peptide exit tunnel. Nucleotides that are divergent between E. coli conformations reported for telithromcyin bound to the ribosome. 23S rRNA and H. marismortui are shown in red. D. radiodurans diverges from E. coli for E. coli is shown in gray. Telithromycin models from H. marismortui (gold), at the 2057-2611 base pair and at nucleotides 752 and 2586. Ribosomal D.radiodurans (cyan), and E. coli (pink) are shown. Nitrogens in the alkyl-aryl RNA helices emanating from this region are marked with dotted lines. arm are also shown for reference.

Dunkle et al. PNAS ∣ October 5, 2010 ∣ vol. 107 ∣ no. 40 ∣ 17153 Downloaded by guest on September 24, 2021 experiments, or the fact that deletion of A752 or mutations in its cleotide mimics of the tRNAs, show that the pyrrolidinyl propyl vicinity lead to resistance (9, 19–22). group, as modeled in the H. marismortui complex, would interfere In the E. coli ribosome, in contrast to H. marismortui or with the positioning of A-site aminoacyl-tRNA (23). Unbiased D. radiodurans, nucleotides A752 and U2609 form a base pair difference electron density maps for clindamycin bound to the that bridges domains II and V in 23S rRNA. Notably, in the struc- E. coli ribosome show density for the propyl group consistent with ture of telithromycin bound to the E. coli ribosome, the alkyl-aryl its position in the H. marismortui structure (Fig. 4 A and B). Thus, arm stacks on the A752-U2609 base pair, a conformation not clindamycin and other lincosamides likely interfere with A-site observed in prior structures (Fig. 2 and Fig. S2A). By contrast, aminoacyl-tRNA binding, as proposed (6). the position of the macrolactone ring remains in essentially The location of the galactose ring, which forms numerous the same conformation observed when telithromycin is bound hydrogen bonds with A2058, A2059, G2505, and A2503 in both to the G2058A H. marismortui 50S subunit (Fig. 2) (6, 13). the G2058A H. marismortui and D. radiodurans 50S subunit struc- The A752-U2609 base pair, which exists in E. coli and many other tures, overlaps with the binding site of the desosamine sugar of eubacteria but not in H. marismortui or D. radiodurans (Fig. S2B), macrolides and ketolides, explaining why lincosamides share re- provides a surface for the entire face of the alkyl-aryl arm to en- sistance mutations with these antibiotic families. In the structure gage in a stacking interaction that likely favors drug binding. of the E. coli ribosome with clindamycin bound, difference elec- Stacking of the alkyl-aryl arm of telithromycin on the A752- tron density for the galactose ring agrees with the prior structural U2609 base pair would also likely lead to protection of A752 from models for placement of the sugar (Fig. 4). The interactions of chemical probes by stabilizing the base pair interaction. To test clindamycin with 23S rRNA nucleotides A2058 and A2503 in this model, we probed E. coli ribosomes in solution by RNA E. coli are identical to those observed with the corresponding footprinting for telithromycin protections of this base pair. We H. marismortui nucleotides. However, nucleotides 2504–2507 also examined Halobacterium halobium (a close relative of are shifted relative to their positions in the G2058A H. marismor- H. marismortui), D. radiodurans and Staphylococcus aureus ribo- tui 50S subunit, leading to alternative interactions with clindamy- somes. Binding of telithromycin to E. coli or S. aureus ribosomes cin (Fig. 4C). The shift probably is due to the presence of A2055 protected A752 from chemical modification (Fig. 3). By contrast, in H. marismortui versus C2055 in E. coli, which stacks against binding of telithromycin to D. radiodurans and H. halobium ribo- U2504 in the structure. This stacking interaction, together with somes did not protect the corresponding nucleotide, although differences in the base pair partners of G2505 and U2609, alters both compounds were bound in all cases (Fig. 3). Given the se- the conformations that nucleotides in this region adopt in E. coli quence conservation of the A752-U2609 base pair among many ribosomes. In the unbiased difference electron density map, a eubacteria, the structural and biochemical data obtained here strong positive peak indicates a shift of G2505 and U2506 toward suggest that the interactions of telithromycin with the E. coli ri- the drug molecule, with U2506 packing perpendicular to the pro- bosome likely reflect those that occur when telithromycin binds pyl group of clindamycin. Additional packing with C2452 leads to the ribosomes of medically relevant eubacterial species. Further- desolvation of the hydrophobic propyl group (Fig. S3). The more, the interaction between the alkyl-aryl arm and the A752- altered position of these nucleotides in E. coli ribosomes relative U2609 base pair helps to explain why deletion of A752 or muta- to their position in H. marismortui 50S subunits allows the brid- tions of U2609 provide resistance to telithromycin (9, 20, 22). ging amine of clindamycin to hydrogen bond to the ribose O4′ of G2505, while preserving contacts with A2058, A2059 and A2503 Clindamycin Sterically Clashes with A-site tRNA. Clindamycin is a (Fig. 4 C and D). Thus, a comparison of the structures of clinda- semisynthetic derivative of the lincosamide class of compounds mycin bound to the G2058A H. marismortui and E. coli ribosomes used clinically to treat gram-positive bacterial infections. Struc- suggests that clindamycin forms a similar number of hydrogen tural models of clindamycin bound to both the G2058A H. mar- bonds to rRNA in the E. coli structure, with increased van der ismortui and D. radiodurans 50S ribosomal subunits (6, 12) agreed Waals contacts with the ribosome. roughly in the placement of the galactose sugar of clindamycin but differed in the positioning of the propyl pyrrolidinyl moiety Chloramphenicol Positioning In and Near the 50S Subunit A-site. Prior of the antibiotic, the portion that is hypothesized to interfere with structural studies identified one binding site for chloramphenicol A-site tRNA positioning (6). In the models of clindamycin bound in the D. radiodurans 50S subunit and a second distinct site in the to the H. marismortui and D. radiodurans 50S subunits, the H. marismortui 50S subunit (12, 15). The differing positions of pyrrolidinyl propyl group is rotated by 180° in one structure re- chloramphenicol are probably due to the fact that wild type lative to the other (Fig. 4A). Structural studies of the ribosome H. marismortui is resistant to chloramphenicol, possibly due to containing aminoacylated A-site and P-site tRNAs, or oligonu- the low affinity of the drug for its primary binding site (10). This

Fig. 3. Telithromycin and erythromycin footprints on 23S rRNA reveal species-specific binding modes. Interactions of antibiotics erythromycin (E) or telithromycin (T) in solution with ribosomes prepared from archaea (H. halobium) or three different bacterial species (D. radiodurans, E. coli,orS. aureus) as revealed by dimethylsulfate (DMS) probing. (Upper) Protections of A2058 and A2059 by both antibiotics to all the tested ribosomes. (Lower) Protection of A752 from DMS modification by telithromycin in E. coli or S. aureus ribosomes, but not in either H. halobium or D. radiodurans ribosomes. Lanes on the gels are labeled as following: A, an A-specific sequencing reaction; K, unmodified ribosome; 0, ribosome modified with DMS in the absence of antibiotics; E and T, ribosome modified with DMS after preincubation with 50 μM of erythromycin or telithromycin, respectively.

17154 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1007988107 Dunkle et al. Downloaded by guest on September 24, 2021 SEE COMMENTARY

Fig. 5. Chloramphenicol bound to the E. coli ribosome. (A) Unbiased differ- F F BIOPHYSICS AND ence electron density ( obs- calc) contoured at 3.5 standard deviations from the mean for chloramphenicol bound to the E. coli ribosome (Left). At right, COMPUTATIONAL BIOLOGY the structure of chloramphenicol in the context of E. coli rRNA is shown (orange) compared to the structure of chloramphenicol reported bound to D. radiodurans ribosomal subunits (cyan). (B) Chloramphenicol (orange) bound to the A-site cleft of the E. coli ribosome. Hydrogen bonds are shown as black dashes. Nucleotides that are sites of resistance mutations to chloramphenicol are labeled in red.

Fig. 4. Clindamycin bound to the E. coli ribosome. (A) The conformations of clindamycin bound to the ribosome. The structural model of clindamycin chloramphenicol in a similar position to the chlorine in clindamy- bound to the D. radiodurans 50S subunit has the pyrrolydinyl propyl group cin (Fig. 4C). The other chlorine atom in chloramphenicol is in a (marked with an asterisk, and showing nitrogen atoms for reference) rotated F F position to contact the exocyclic amine of A2062, a residue that by 180 degrees. (B) Unbiased difference electron density ( obs- calc) for clindamycin bound to the E. coli ribosome. The electron density is contoured at when mutated results in chloramphenicol resistance (10). 3.5 standard deviations from the mean. (C) E. coli 23S rRNA is superpositioned Finally, the amine of chloramphenicol hydrogen bonds to the with H. marismortui 23S rRNA to reveal that some nucleotides (2504–2507) nonbridging phosphate oxygen of G2505 (Fig. 5B). are shifted in space due to the sequence difference at 2055, C in E. coli, Because rRNA sequences in the region of chloramphenicol AinH. marismortui. E. coli nucleotides are in gray, H. marismortui in yellow. binding are conserved in both E. coli and D. radiodurans, se- (D) A diagram of the interactions of clindamycin with the H. marismortui (Left) and E. coli (Right) ribosomes. Dashed lines represent hydrogen bonds, quence divergence cannot explain the different orientations of and curves represent key van der Waals interactions. the antibiotic. Unless the second shell rRNA residues dramati- cally affect chloramphenicol binding, there is likely one principal intrinsic tolerance of archaea to chloramphenicol is likely due to mode of interaction between chloramphenicol and eubacterial rRNA sequence differences between archaea and bacteria; i.e. in ribosomes (25). The unbiased difference electron density in nucleotides C2055 of H73, and U2609, and C2610 adjacent to the the E. coli structure determination provides a much clearer view base of rRNA helix H73 (Fig. 1C). of details of chloramphenicol orientation than is provided by the In the structure of chloramphenicol bound to the E. coli ribo- electron density used to model chloramphenicol bound to the D. some, we did not observe any difference electron density in the radiodurans 50S subunit (compare Fig. 5A with Fig. 1C of ref. 12). chloramphenicol binding site identified in the H. marismortui 50S Furthermore, the structural model of chloramphenicol bound subunit (24). By contrast, clear positive difference electron den- to the E. coli ribosome is at higher resolution and with a lower sity is located at the site of chloramphenicol binding reported in R value (25%) when compared to the D. radiodurans model the D. radiodurans 50S subunit, but this density is not entirely con- free (R ¼ 32%)(Table S1). Finally, the interactions between chlor- sistent with the reported orientation of chloramphenicol (Fig. 5 A free amphenicol and rRNA in the orientation in the present model and B) (12). In the E. coli ribosome complex, the nitrobenzene moiety of chloramphenicol overlaps with the position that would are more chemically favorable due to additional stacking interac- be occupied by the pyrrolidinyl propyl group of clindamycin tions that should facilitate drug binding. Notably, in the present (Figs. 1B,4C, and 5B). When compared to the structure of structural model of chloramphenicol bound to the E. coli 70S clindamycin bound to the E. coli ribosome, the base of U2506 is ribosome, the nitrobenzene ring stacks on C2452 in the same rotated such that the hydrophilic nitro group in chloramphenicol orientation that the methoxyphenyl ring of the related compound points into solvent. The nitrobenzene ring of chloramphenicol is anisomycin stacks on C2452 when bound to the H. marismortui stacked on C2452 (Fig. 5B) and positions one chlorine atom of 50S subunit (26).

Dunkle et al. PNAS ∣ October 5, 2010 ∣ vol. 107 ∣ no. 40 ∣ 17155 Downloaded by guest on September 24, 2021 Discussion tional phylogenetic differences contribute to resistance (36). The rRNA of the PTC is highly conserved throughout the 3 Taken together with phylogenetic data, the present structural domains of life, explaining why many antibiotics that bind in and biochemical data suggest that, in addition to position or near the PTC can inhibit a wide range of species. However, 2058, divergence of rRNA at positions 752, 2609, 2610, and many antibiotics that bind in this region are selective. For exam- 2055 also contribute to the species specificity of PTC and peptide ple erythromycin does not bind to eukaryotic or archaeal ribo- elongation inhibitors. The availability of high-resolution struc- somes except at extremely high concentrations. The identity of tures of the ribosome and ribosomal subunits from archaea nucleotide 2058, adenosine in eubacteria, guanosine in eukar- and divergent bacteria will now make it possible to probe these yotes and archaea, is thought to be a major contributor to this and other sequence determinants of PTC and peptide elongation phenomenon (6). Conversely, wild type eubacteria are resistant inhibitors. to anisomycin whereas archaea and eukaryotes are sensitive (26, 27). Experimental Procedures Our data reveal several rRNA residues that can account for Ribosome Purification, Crystallization, and Antibiotic Soaking Experi- the selectivity of antibiotic action. In the E. coli ribosome, 23S ments. Ribosomes were purified from MRE600 E. coli cells as rRNA residues A752 and U2609 form a base pair that interacts described previously (37). Ribosome crystals were grown as with a key element of telithromycin, its extended 11,12 alkyl-aryl described previously (38). For antibiotic soaking experiments, arm (Fig. 3 A and B). Our data suggest that disruption of this base the cryoprotection buffer was supplemented with 48 μM of teli- pair would decrease the affinity of telithromycin for the ribosome, thromcyin or 100 μM of either erythromycin, chloramphenicol or consistent with mutation U2609C or deletion of A752 that lead to clindamycin. Erythromycin, chloramphenicol and clindamycin low level telithromycin resistance (20, 22). In D. radiodurans and were obtained from Sigma. Telithromycin was a gift from Cempra H. marismortui, this base pair does not exist, leading to a different Pharmaceuticals. The telithromycin stock solution in 50% acetic conformation of the alkyl-aryl side chain (6, 13) and likely acid was diluted ∼800-fold in cryoprotection buffer for soaks. decreasing telithromycin affinity for the ribosome (Fig. 4). Erythromycin and chloramphenicol stock solutions were made in Nucleotide C2055 in eubacteria is an adenosine in archaea and ethanol, and diluted 100-fold to working concentrations in cryo- eukaryotes (Table S2) (28), and also seems to serve as an impor- protection buffer. Clindamycin was dissolved in DMSO and di- tant determinant for the spectrum of action of A-site inhibitors luted 100× in cryoprotection buffer for soaking experiments. such as chloramphenicol and clindamycin, as well as other anti- In all four cases incubation of the crystals with antibiotic took biotics (29, 30). Our structures reveal that alteration of A2055, place overnight. Crystals were then flash frozen in liquid nitrogen. which stacks on U2504 in the H. marismortui 50S subunit structures, to C2055 in E. coli leads to a displacement of U2504 along X-ray Diffraction Experiments, Model Building, and Figure Prepara- with G2505, U2506, and C2507 (Figs. 1B and 4C). Thus the se- tion. X-ray diffraction data were measured at beamlines 8.3.1 quence difference at C2055 results in a change in the position of and 12.3.1 of the Advanced Light Source, Lawrence Berkeley four universally conserved PTC nucleotides (TableS2). The struc- National Laboratory, using 0.1–0.3 ° oscillations at 100 K and tures of the E. coli ribosome show that chloramphenicol and clin- recorded on an ADSC Q315 detector. X-ray reflections were re- damycin both interact with these nucleotides. G2505 hydrogen duced and scaled using HKL2000. Difference electron density bonds to clindamycin whereas U2506 desolvates the propyl group (F -F ) maps were calculated using the phenix.refine module of clindamycin (Fig. 4 C and D). G2505 forms hydrogen bonds to obs calc of the PHENIX software suite (39). Antibiotics were positioned chloramphenicol, interactions that are not possible in H. maris- based on the unbiased difference electron density obtained in mortui, given the conformation of this residue in H. marismortui each experiment. If necessary, changes in the coordinates for ribosomal subunits (Fig. 4B). These structural data explain why rRNA nucleotides dictated by the difference electron density archaeal and eukaryotic ribosomes bind lincosamides and chloramphenicol poorly. In fact, difference electron density for chlor- were made using Coot (40) and phenix.refine was used for posi- amphenicol is not observed in the eubacterial binding site even tional and atomic displacement parameter (ADP) refinement of when H. marismortui ribosomal subunits are soaked in millimolar the model. The crystals contain two copies of the ribosome in the concentrations of the compound (15). In addition, the mutation asymmetric unit, with molecule 1 (PDB coordinates 3OAT, C2055A in eubacterial ribosomes increases the minimal inhibi- 3OFR, 3OFC, 3OFZ for the 50S subunit in complex with telithro- tory concentration (MIC) of chloramphenicol and clindamycin mycin, erythromycin, chloramphenicol, and clindamycin, respec- (31). Furthermore, the C2055A mutation in M. smegmatis ribo- tively) exhibiting lower ADP values and clearer electron density somes leads to resistance to pleuromutilin antibiotics, which also than molecule 2. Whereas difference electron density for antibio- bind the eubacterial PTC (32). tics was observed in both molecule 1 and molecule 2 of the crystal The proposed role of 23S rRNA nucleotide 2055 as an anti- in each experiment, coordinates for antibiotics were only mod- biotic resistance spectrum determinant through its interaction eled into molecule 1. Ribosomal RNA sequence alignments were with A-site cleft nucleotides 2504 and 2506–2507 suggest that made using MUSCLE (41) or obtained from the Comparative mutations of these residues should also appear in mutants resis- RNA Website (28). Coordinate superpositions were performed tant to chloramphenicol and lincosamides. Despite the sensitive in PyMOL using the C1′ atoms of domain V of 23S rRNA, location of these nucleotides in the PTC and their universal excluding regions where insertions or deletions occur. Figures conservation, mutations of nucleotide 2504 confer resistance were made using PyMOL (42). to chloramphenicol (31, 33) and increase the MIC of clindamcyin (31). The lack of posttranscriptional modification (pseudouridi- Preparation of Ribosomes and Ribosomal Subunits for RNA Probing. nylation) at this position renders bacteria hypersensitive to Ribosomes and large ribosomal subunits were isolated from several peptidyl transferase inhibitors (34). E. coli (strain JM109) or S. aureus (strain RN4220) using stan- Mutation of G2058A in archaeal ribosomes leads to a ∼104- dard protocols described in ref. 43. Large ribosomal subunits fold improvement in macrolide binding to H. marismortui or prepared from D. radiodurans were a gift of Francheschi and H. halobium 50S subunits, suggesting that the identity of a single Skripkin (Rib-X, Inc). To increase affinity of macrolides for nucleotide could explain the insensitivity of archaeal and eukar- the archaeal large ribosomal subunits, G2058 in the 23S rRNA yotic ribosomes toward macrolides (6, 35). However, in S. cere- gene (E. coli numbering) of the single ribosomal operon of visiae ribosomes carrying the G2058A mutation in their ribosomal H. halobium was mutated to A. This mutation was introduced RNA were still not inhibited by erythromycin, suggesting addi- together with the selective anisomycin resistance mutation

17156 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1007988107 Dunkle et al. Downloaded by guest on September 24, 2021 C2452U using the procedure described in ref. 44. Haloarchaeal with dimethylsulfate and the distribution of modified residues

ribosomes were isolated as previously described (45). was analyzed by primer extension. SEE COMMENTARY

Chemical Probing of the Interaction of Erythromycin and Telithromy- ACKNOWLEDGMENTS. We thank K. Frankel, S. Classen, and G. Meigs for help cin with Large Ribosomal Subunits. Chemical probing of interac- with data measurement at the SIBYLS and 8.3.1 beamlines at the Advanced Light Source; P. Afonine and P. Adams for help with Phenix refinement; and tions of erythromycin and telithromycin with bacterial and S.-H. Kim, P. Adams, and J. Holton for useful crystallographic discussions. haloarchaeal large ribosomal subunits was carried out using pro- This work was funded by the National Institutes of Health Grant cedures described in ref. 46 and in ref. 47 with ribosomes and GM65050 (J.H.D.C.) and National Cancer Institute Grant CA92584 for the μ SIBYLS and 8.3.1 beamlines, by the National Science Foundation Grant drugs present at 200 nM and 50 M, respectively. Ribosomal MCB-0824739 (A.S.M.), and by the Department of Energy Grant DE-AC03 subunits, vacant or complexed with antibiotics, were modified 76SF00098 for the SIBYLS and 8.3.1 beamlines.

1. Cundcliffe E (2000) Antibiotic biosynthesis: Some thoughts on “why” and “how”. 25. Nierhaus D, Nierhaus KH (1973) Identification of the chloramphenicol-binding The Ribosome:Structure, Function, Antibiotics and Cellular Interactions, eds RA Gar- protein in Escherichia coli ribosomes by partial reconstitution. Proc Natl Acad Sci rett, A Liljas, AT Matheson, PB Moore, and HF Noller (ASM Press, Washington, DC), USA 70:2224–2228. pp 409–417. 26. Blaha G, Gurel G, Schroeder SJ, Moore PB, Steitz TA (2008) Mutations outside the 2. Sohmen D, Harms JM, Schluenzen F, Wilson DN (2009) SnapShot: Antibiotic inhibition anisomycin-binding site can make ribosomes drug-resistant. J Mol Biol 379:505–519. of protein synthesis II. Cell 139 212.e211. 27. Hummel H, Böck A (1987) 23S ribosomal RNA mutations in halobacteria conferring 3. Polacek N, Mankin AS (2005) The ribosomal peptidyl transferase center: Structure, resistance to the anti-80S ribosome targeted antibiotic anisomycin. Nucleic Acids function, evolution, inhibition. Crit Rev Biochem Mol Biol 40:285–311. Res 15:2431–2443. 4. Vester B, Douthwaite S (2001) Macrolide resistance conferred by base substitutions in 28. Cannone JJ, et al. (2002) The comparative RNA web (CRW) site: An online database of 23S rRNA. Antimicrob Agents Chemother 45:1–12. comparative sequence and structure information for ribosomal, intron, and other 5. Mankin AS (2008) Macrolide myths. Curr Opin Microbiol 11:414–421. RNAs. BMC Bioinformatics 3:2. 6. Tu D, Blaha G, Moore PB, Steitz TA (2005) Structures of MLSBK antibiotics bound 29. Davidovich C, et al. (2007) Induced-fit tightens pleuromutilins binding to ribosomes to mutated large ribosomal subunits provide a structural explanation for resistance. and remote interactions enable their selectivity. Proc Natl Acad Sci USA – Cell 121:257–270. 104:4291 4296. 7. Poehlsgaard J, Douthwaite S (2005) The bacterial ribosome as a target for antibiotics. 30. Gürel G, Blaha G, Moore PB, Steitz TA (2009) U2504 determines the species specificity Nat Rev Microbiol 3:870–881. of the A-site cleft antibiotics: the structures of tiamulin, homoharringtonine, and – 8. Ettayebi M, Prasad SM, Morgan EA (1985) Chloramphenicol-erythromycin resistance bruceantin bound to the ribosome. J Mol Biol 389:146 156. mutations in a 23S rRNA gene of Escherichia coli. J Bacteriol 162:551–557. 31. Long KS, et al. (2009) Single 23S rRNA mutations at the ribosomal peptidyl transferase 9. Canu A, et al. (2002) Diversity of ribosomal mutations conferring resistance to macro- centre confer resistance to valnemulin and other antibiotics in Mycobacterium smeg- – lides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae. matis by perturbation of the drug binding pocket. Mol Microbiology 71:1218 1227. BIOPHYSICS AND Antimicrob Agents Chemother 46:125–131. 32. Schlünzen F, Pyetan E, Fucini P, Yonath A, Harms JM (2004) Inhibition of peptide

10. Mankin AS, Garrett RA (1991) Chloramphenicol resistance mutations in the single bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from COMPUTATIONAL BIOLOGY Deinococcus radiodurans in complex with tiamulin. Mol Microbiol 54:1287–1294. 23S ribosomal RNA gene of the archaeon Halobacterium halobium. J Bacteriol 33. Blanc H, Wright CT, Bibb MJ, Wallace DC, Clayton DA (1981) Mitochondrial DNA of 173:3559–3563. chloramphenicol resistant mouse cells contains a single nucleotide change in the 11. Wilson DN, Harms JM, Nierhaus KH, Schlunzen F, Fucini P (2005) Species-specific region encoding the 3′ end of the large ribosomal RNA. Proc Natl Acad Sci USA antibiotic–ribosome interactions: Implications for drug development. Biol Chem 78:3789–3793. 386:1239–1252. 34. Toh SM, Mankin AS (2008) An indigenous posttranscriptional modification in the 12. Schluenzen F, et al. (2001) Structural basis for the interaction of antibiotics with the ribosomal peptidyl transferase center confers resistance to an array of protein peptidyl transferase centre in eubacteria. Nature 413:814–821. synthesis inhibitors. J Mol Biol 380:593–597. 13. Berisio R, et al. (2003) Structural insight into the antibiotic action of telithromycin 35. Böttger EC, Springer B, Prammananan T, Kidan Y, Sander P (2001) Structural basis for against resistant mutants. J Bacteriol 185:4276–4279. selectivity and toxicity of ribosomal antibiotics. EMBO Rep 2:318–323. 14. Hansen JL, et al. (2002) The structures of four macrolide antibiotics bound to the large 36. Bommakanti AS, Lindahl L, Zengel JM (2008) Mutation from guanine to adenine in 25S ribosomal subunit. Mol Cell 10:117–128. rRNA at the position equivalent to E. coli A2058 does not confer erythromycin 15. Hansen JL, Moore PB, Steitz TA (2003) Structures of five antibiotics bound at the sensitivity in Sacchromyces cerevisae. RNA 14:460–464. peptidyl transferase center of the large ribosomal subunit. J Mol Biol 330:1061–1075. 37. Schuwirth BS, et al. (2005) Structures of the bacterial ribosome at 3.5 A resolution. 16. Stephenson G, Stowell J, Toma P, Pfeiffer R, Byrn S (1997) Solid-state investigations of Science 310:827–834. erythromycin A dihydrate: Structure, NMR spectroscopy, and hygroscopicity. J Pharm 38. Zhang W, Dunkle JA, Cate JH (2009) Structures of the ribosome in intermediate – Sci 86:1239 1244. states of ratcheting. Science 325:1014–1017. 17. Vazquez-Laslop N, Thum C, Mankin AS (2008) Molecular mechanism of drug- 39. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro- – dependent ribosome stalling. Mol Cell 30:190 202. molecular structure solution. Acta Crystallogr D 66:213–221. 18. Hansen LH, Mauvais P, Douthwaite S (1999) The macrolide-ketolide antibiotic binding 40. Emsley P, Cowtan K (2004) Coot: Model-building tools for molecular graphics. Acta site is formed by structures in domains II and V of 23S ribosomal RNA. Mol Microbiol Crystallogr D 60:2126–2132. – 31:623 631. 41. Edgar RC (2004) MUSCLE: Multiple sequence alignment with high accuracy and high 19. Xiong L, Shah S, Mauvais P, Mankin AS (1999) A ketolide resistance mutation in domain throughput. Nucleic Acids Res 32:1792–1797. II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre. 42. Delano WL The PyMOL User’s Manual (DeLano Scientific, San Carlos, CA). – Mol Microbiol 31:633 639. 43. Moazed D, Noller HF (1989) Interaction of tRNA with 23S rRNA in the ribosomal A, 20. Garza-Ramos G, Xiong L, Zhong P, Mankin A (2001) Binding site of macrolide antibio- P, and E sites. Cell 57:585–597. tics on the ribosome: New resistance mutation identifies a specific interaction of 44. Mankin AS, Zyrianova IM, Kagramanova VK, Garrett RA (1992) Introducing mutations ketolides with rRNA. J Bacteriol 183:6898–6907. into the single-copy chromosomal 23S rRNA gene of the archaeon Halobacterium 21. Xiong L, Korkhin Y, Mankin AS (2005) Binding site of the bridged macrolides in the halobium by using an rRNA operon-based transformation system. Proc Natl Acad Escherichia coli ribosome. Antimicrob Agents Chemother 49:281–288. Sci USA 89:6535–6539. 22. Novotny GW, Jakobsen L, Andersen NM, Poehlsgaard J, Douthwaite S (2004) Ketolide 45. Tan GT, DeBlasio A, Mankin AS (1996) Mutations in the peptidyl transferase center of antimicrobial activity persists after disruption of interactions with domain II of 23S 23S rRNA reveal the site of action of sparsomycin, a universal inhibitor of translation. rRNA. Antimicrob Agents Chemother 48:3677–3683. J Mol Biol 261:222–230. 23. Voorhees RM, Weixlbaumer A, Loakes D, Kelley AC, Ramakrishnan V (2009) Insights 46. Merryman C, Noller HF (1998) Footprinting and modification-interference analysis of into substrate stabilization from snapshots of the peptidyl transferase center of the binding sites on RNA. RNA:Protein Interactions, A Practical Approach, ed CWJ Smith intact 70S ribosome. Nat Struct Mol Biol 16:528–533. (Oxford University Press, Oxford). 24. Hansen JL, Moore PB, Steitz TA (2003) Structures of five antibiotics bound at the 47. Mankin AS (1997) Pactamycin resistance mutations in functional sites of 16S rRNA. peptidyl transferase center of the large ribosomal subunit. J Mol Biol 330:1061–1075. J Mol Biol 274:8–15.

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