Macrolide Antibiotics in the Ribosome Exit Tunnel: Speciesspecific Binding and Action

Ann. N.Y. Acad. Sci. ISSN 0077-8923

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Antimicrobial Therapeutics Reviews

Macrolide antibiotics in the ribosome exit tunnel: species-speciﬁc binding and action

Krishna Kannan and Alexander S. Mankin Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, Illinois

Address for correspondence: Dr. Alexander Mankin, Center for Pharmaceutical Biotechnology-m/c 870, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607. [email protected]

Macrolide antibiotics bind in the nascent peptide exit tunnel of the ribosome and inhibit protein synthesis. The majority of information on the principles of binding and action of these antibiotics comes from studies that employed model organisms. However, there is a growing understanding that the binding of macrolides to their target, as well as the mode of inhibition of translation, can be strongly influenced by variations in ribosome structure between bacterial species. Awareness of the existence of species-specific differences in drug action and appreciation of the extent of these differences can stimulate future work on developing better macrolide drugs. In this review, representative cases illustrating the organism-specific binding and action of macrolide antibiotics, as well as species-specific mechanisms of resistance are analyzed.

Keywords: macrolide; ribosome; protein synthesis; nascent peptide exit tunnel; posttranscriptional modiﬁcations

ing drugs such as clarithromycin, roxithromycin, Introduction and azithromycin (Fig. 1A and B), which be- Macrolides are an important class of antibiotics ef- long to the second generation of macrolides, ex- fective against a number of pathogenic bacteria. hibit increased acid stability, better oral bioavail- These mostly bacteriostatic drugs are commonly ability, improved pharmacodynamics, and broader used to treat respiratory tract infections includ- antimicrobial spectrum. The second-generation ing community acquired pneumonia, pharyngitis, macrolides showed particularly improved activ- and tonsillitis, along with skin and soft tissue in- ity against Haemophilus influenzae and intracel- fections, urogenital infections, and orodental in- lular pathogens such as Mycobacterium avium- fections. Macrolides were introduced into medi- intracellulare. The emergence and broad spread cal practice almost 60 years ago and continue to of resistance prompted the development of the be viewed as excellent antibiotics with high po- new generation of macrolides. Ketolides (Fig. 1C), tency and low toxicity. Erythromycin A, a nat- representing the third generation, show improved ural product of Saccharopolyspora erythraea,was potency against many sensitive and some resis- the first macrolide to be advanced to medical use tant strains and are often associated with bacte- in the early 1950s for the treatment of bacte- ricidal activity. Macrolide antibiotics with an ex- rial infections.1 Erythromycin (Fig. 1A) is effective tended macrolactone ring, such as 16-membered against many Gram-positive pathogenic bacteria in- macrolides (Fig. 1D), find extensive use in veterinary cluding Staphylococcus aureus, Streptococcus pneu- medicine and are sometimes also used in humans. moniae, S. pyogenes,andEnterococcus sp.;some Macrolides inhibit bacterial growth by binding to Gram-negative bacteria such as Neisseria gonor- the ribosome and blocking protein synthesis. The ri- rhoeae, Bordetella pertussis,andHaemophilus in- bosome is an RNA-based macromolecular machine fluenzae; and intracellular pathogens such as My- withamolecularweightofca.2.5millionDa(inbac- coplasma sp., Legionella sp.,andChlamydia sp. teria). It represents one of the best cellular antibiotic Semisynthetic derivatives of erythromycin, includ- targets. The ribosome is composed of two subunits, doi: 10.1111/j.1749-6632.2011.06315.x Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. 33 Species-specific binding and action of macrolides Kannan & Mankin

Figure 1. Chemical structures of representative macrolide antibiotics. The numbering of the macrolactone atoms is illustrated on the erythromycin structure. Side chains discussed in the review are labeled. small and large (also known as the 30S and 50S Most of what is known about the interactions subunits, respectively, based on their sedimentation of protein synthesis inhibitors with their targets coefﬁcients). The decoding center in the small ribo- comes from the studies of ribosomes from selected somal subunit is responsible for the selection of the model organisms. Powerful biochemical techniques aminoacyl-tRNAs in based on the order of codons have been used to locate drug binding sites in Es- in mRNA. The amino acids are assembled into a cherichia coli ribosomes.5–8 Additional information polypeptide chain in the peptidyl transferase center about drug–ribosome interactions came from ge- (PTC) located in the large ribosomal subunit. The netic studies that were employed to identify re- newly assembled polypeptides leave the ribosome sistance mutations in E. coli ribosomal proteins through the nascent peptide exit tunnel (NPET), and rRNA.9–14 These studies were later expanded which starts at the PTC and spans the body of the and enhanced by including organisms with sim- large subunit2 (Fig. 2A and 2B). Macrolides bind in pler rRNA genetics, such as the halophilic archeon the NPET close to the PTC. They hinder the passage Halobacterium halobium15,16 and engineered strains of the newly synthesized polypeptides through the of Mycobacterium smegmatis,17 which carry sin- tunnel, thereby interrupting translation elongation, gle rDNA alleles. Subsequent advent of ribosome most commonly at the early rounds of protein syn- crystallography resulted in atomic-level models of thesis.3 In addition, some macrolides with extended ribosome-drug complexes.18–21 However, since only side chains reach close to the catalytic center and a few types of ribosomes are amenable to crystalliza- interfere with peptide bond formation.4 tion, the available complexes have been produced

34 Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. Kannan & Mankin Species-speciﬁc binding and action of macrolides

Figure 2. Binding site of macrolide antibiotics in the ribosome. (A) Macrolide antibiotics (e.g., erythromycin) partially obstruct the exit tunnel. View down NPET from the interface side of the large ribosomal subunit. The erythromycin molecule is shown in salmon. (B) Erythromycin binds in the exit tunnel close to the PTC. Small (30S) and large (50S) ribosomal subunits are colored yellow and blue, respectively. The surface of the exit tunnel is shown in grey and peptidyl-tRNA with a short nascent peptide is green. The atoms of the erythromycin molecule are shown as salmon-colored spheres. (C) rRNA residues in the erythromycin binding site in the E. coli ribosome (PDB accession number 3OFR).21 Erythromycin molecule is shown as sticks-and-surface representation. The 23S rRNA residues A2451 and C2452 located in the PTC A-site are shown for orientation. (D) The mycaminose-mycarose- isovalerate side chain of carbomycin A can reach into the PTC (H. marismortui 50S subunit, PDB accession number 1K8A).19 (E) The mycinose side-chain of tylosin stretches towards the loop of helix 35 and comes into a close contact with rRNA residues A752 and G748 and ribosomal proteins L4 and L22 (H. marismortui 50S subunit, PDB accession number 1K9M).19 by using the ribosomes and ribosomal subunits Most of the aforementioned experimental mod- from a very limited set of species—the halophilic els used in biochemical, genetic, or crystallographic archeon Haloarcula marismortui, the radiation- studies are only distantly related to clinically relevant resistant Gram-positive Deinococcus radiodurans, pathogens. Because the ribosome structure is highly the Gram-negative mesophilic E. coli, and the ther- conserved, results obtained with model organisms mophilic Thermus thermophilus. are often directly and sometimes indiscriminately

Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. 35 Species-specific binding and action of macrolides Kannan & Mankin extrapolated to pathogenic bacterial species. How- composed predominantly of rRNA nucleotides be- ever, in many instances, the species-specific idiosyn- longing to domains II and V of the 23S rRNA. The crasies in the ribosome structure result in important central macrolactone ring of the drug establishes differences in drug binding and action. hydrophobic interactions with the rRNA residues In this short review, we will first provide a general 2057, 2611, and 2058 (E. coli numbering of rRNA overview of the mode of macrolide binding to the nucleotides is used throughout) that form the tun- ribosome and the current view of the mechanism of nel wall on the side of the PTC A site. In most of drug action. We will then discuss several examples the available high-resolution crystallographic struc- demonstrating that the binding of macrolides, and tures, the macrolactone is positioned flat against the thus the mode of their action, can vary substantially wall with the side chains protruding either up the in different species. tunnel, toward the PTC active site, or down, toward the tunnel constriction. The C5 amino sugar Chemical structure of macrolide antibiotics (desosamine in 14- and 15-member ring macrolides Clinically useful macrolides are characterized by the or mycaminose of the mycarose-mycaminose disac- presence of a 14-, 15-, or 16-atom macrolactone charide in the 16-member ring macrolides) extends ring, to which several neutral or amino sugars and in the direction of the PTC and is drawn close to the other side chains are attached (Fig. 1). The proto- crevice between the bases of the adenine residues typical natural macrolide antibiotic, erythromycin, A2058 and A2059. A hydrogen bond donated by is composed of a 14-member lactone ring that car- the 2-OH of the desosamine to the N1 of A2058 ries two sugars, cladinose, and desosamine, attached additionally contributes to drug binding. The inter- at positions C3 and C5, respectively (Fig. 1A). In the actions of the C5 side chain with A2058 and A2059 semisynthetic azithromycin (Fig. 1B), the macrolac- are very important: dimethylation of the exocyclic tone ring of erythromycin is extended by an addi- amine of A2058 by erythromycin resistance methyl- tional nitrogen atom. The 16-membered macrolides transferases (Erms) or mutations of A2058 or A2059 like tylosin or carbomycin contain an extended dramatically reduce the affinity of all macrolides for disaccharide at the C5 position and often possess the ribosome.14,24 While the C5 desosamine of the several additional side chains attached at other po- 14- and 15-member ring macrolides does not reach sitions of the macrolactone (Fig. 1D). In the third- the PTC, the longer disaccharide C5 side chains of generation macrolides, the ketolides, a keto group 16-member ring macrolides approaches the PTC replaces the C3 cladinose of erythromycin (Fig. 1C). active site more closely and can affect the cataly- In addition, ketolides carry a 11,12 cyclic carbamate sis of peptide bond formation. The C5 disaccharide and an extended alkyl–aryl side chain that can be in carbomycin and josamycin is further extended by linked at different sites to the macrolactone moiety. an isovalerate moiety (Figs. 1D and 2D) that reaches Some clinically promising ketolides are additionally directly into the PTC A site, where it is positioned fluorinated at C2.22,23 in the hydrophobic crevice formed by residues Both the macrolactone ring and the side chains A2451 and C2452 in the heart of the catalytic of macrolide antibiotics contribute to the binding center.4,19 affinity of the drug to the ribosomal target. How- The cladinose sugar attached at C3 of ery- ever, while variation in the structure of the central thromycin and second-generation macrolides macrolactone ring has little influence on the mode comes into close contact with C2610 and G2505 of drug binding or inhibition of translation,19,21 the (Fig. 2C).19,21 Although the C2610 mutation has lit- structure of the side chains directly affects the inter- tle effect upon drug binding or susceptibility, this action of the drug with specific rRNA residues, the nucleotide is apparently important for fine-tuning mechanism of macrolide action, and its propensity interactions among the drug, the ribosome, and to activate resistance mechanisms. the nascent peptide because the C2610U mutation affects the ability of the cladinose-containing Macrolide-binding site in the ribosome macrolides to activate the expression of drug- Macrolides bind in the upper chamber of the NPET, inducible erm genes.25 between the PTC and the constriction formed by The C3 cladinose is absent in ketolides and 16- proteins L4 and L22 (Fig. 2B). The binding site is member ring macrolides. However, the presence of

36 Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. Kannan & Mankin Species-specific binding and action of macrolides an extended alkyl–aryl side chain in ketolides or ad- ample, the C14-linked mycinose of tylosin directly ditional side chains in 16-member ring macrolides interacts with protein L22 (Fig. 2E), whereas the compensate for the absence of cladinose, because C9-linked forosamine of spiramycin reaches close they increase the drug’s affinity by establishing ad- to L4.19 Mutations in these proteins confer resis- ditional interactions with nucleotides of domain II tance to various macrolides. However, the effects of of the 23S rRNA. Recent crystallographic structures ribosomal protein mutations upon binding of most of telithromycin, the first clinically approved ke- macrolide antibiotics could be either allosteric or tolide, complexed to the E. coli or T. thermophilus invoke unconventional mechanisms.29,30 The struc- ribosomes confirmed the previously proposed close ture of the ribosomal proteins is less conserved than contacts of the side chain with the loop of helix that of the rRNA. Variation in protein structure 35.20,21 Similarly, the C14-linked mycinose of tylosin may contribute to the ribosome-specific binding of interacts with bases at positions 748, 751, and 752 in macrolides. the helix 35 loop19 (Fig. 2E). The ribosomal contacts Mechanism of action of mycinose are clearly important for tylosin binding, because when these interactions are abolished According to the commonly accepted model, by the methylation of A748 by RlmAII methyltrans- macrolide antibiotics inhibit protein synthesis by ferase (in conjunction with the A2058 monomethy- obstructing the growth of the nascent peptide chain. lation), tylosin fails to inhibit cell growth.26 The 14- and 15-atom lactone ring macrolides have Some 16-membered macrolides (e.g., tylosin) little effect during the early rounds of translation. carry an ethyl-aldehyde group at the C6 position. Only after the first few amino acids are polymer- In the crystallographic structures of these drugs ized and the growing polypeptide reaches the site complexed with the H. marismortui 50S subunit, a of drug binding does the subsequent progression continuous electron density connects the aldehyde of the peptide through the NPET become inhib- group to the exocyclic amine of A2062, which is ited, and peptidyl-tRNA dissociates from the ribo- compatible with the formation of a reversible cova- some.31,32 With the cladinose containing 14- and lent bond.19 A similar covalent bond was observed 15-membered macrolides bound in the tunnel, the between josamycin and A2062 in the D. radiodu- dissociated peptidyl-tRNAs carry a 6–9 amino acid– rans 50S subunit.27 Abolishing the formation of long peptide.33 Ketolides, which lack the C3 cladi- this putative bond is associated with decreased drug nose, allow polymerization of 9–10 amino acids.33 affinity and reduced ability of the drug to inhibit Accumulation of peptidyl-tRNA, leading to the ex- translation.28 haustion of the pools of free tRNAs in the cell, Some of the nucleotides that form the macrolide- could be the major cause of translation cessation binding site, for example, the 2057–2611 base pair or in macrolide-treated cells.32 the loop of helix 35, exhibit considerable variation When the drug obstructs the growth of the among bacterial species. However, even the place- nascent peptide, the peptidyl-tRNA drop-off rate ment of conserved rRNA residues involved in drug and the spontaneous dissociation rate of the drug binding may vary in different ribosomes because from the ribosome have important consequences.34 of the species-specific interactions with the non- If the antibiotic dissociates prior to the peptidyl- conserved second-shell nucleotides that buttress the tRNA drop-off and a few amino acids are added to drug binding site components. Therefore, it is not the polypeptide before a new antibiotic molecule so surprising that the exact orientation of the same binds, then the synthesis of the protein will be macrolide molecule bound to ribosomes of different carried to its completion because longer nascent species can exhibit substantial variation (see below). polypeptides prevent drug rebinding.35 Because of Although most of the interactions that macrolides that, some residual protein synthesis can be ob- establish with the ribosome involve rRNA, long side served even when cells are treated with a high chains of some 16-member ring macrolides and the concentration of drugs. The dissociation rate of ketolides extend far enough down the tunnel to peptidyl-tRNA depends on the nascent peptide come into direct contact with the ribosomal pro- sequence, length, and its interaction with the NPET, teins L4 and L22, which form the constriction of whereas the dissociation rate of the drug is deter- the NPET ca. 20–35 A˚ away from the PTC.2 For ex- mined by its structure and the specifics of its contacts

Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. 37 Species-specific binding and action of macrolides Kannan & Mankin with the NPET in the drug binding site.36 There- Species-specific macrolide–ribosome fore, the residual protein synthesis afforded during interactions macrolide treatment can be protein specific,28 and The resolution of the currently available structures thus, species specific. Furthermore, some nascent of ribosome–macrolide complexes range from 2.5 A˚ peptides have the capacity to evict the drug from the , to 3.5 A,˚ which makes it possible to examine drug– ribosome,37 38 whereas others can thread through target interactions at an atomic level. However, it the tunnel obstructed by the macrolide antibiotic39 is important to remember that the X-ray structures (Kannan et al., in preparation). These aspects may need to be treated with a certain degree of discre- contribute to the idiosyncratic action of macrolide tion. While the binding and the action of macrolide antibiotics against different bacterial species. antibiotics are likely to be influenced by the nascent In contrast to 14- and 15-member ring macrolides peptide, none of the structures solved to date contain that do not inhibit polymerization of the first few peptidyl–tRNA. Therefore, these structures reflect amino acids, the 16-membered macrolides that the initial binding mode of the antibiotic to the va- carry a disaccharide (mycaminose–mycarose) side cant ribosome, rather than when the drug elicits its chain at the C5 position can block translation at ear- inhibitory action on translation. Furthermore, crys- lier stages. The mycaminose–mycarose chain that tallization conditions significantly deviate from the reaches into the PTC can directly interfere with environment in which the ribosome exists in the cell the formation of the second or even first peptide cytoplasm, which may influence the conformation bonds.4 In carbomycin A and josamycin, the disac- of the ribosome, its dynamics, and its interactions charide molecule is further extended by isovalerate with antibiotics. In addition, as mentioned earlier, appended to the mycarose sugar. This group reaches none of the published structures employ ribosomes into the amino acid binding pocket of the PTC A- from organisms closely related to pathogenic Gram- site. Therefore, these drugs can affect peptide bond positive bacteria. formation in an amino acid-specific manner. Thus, Although the available structures converge in in- josamycin diminishes the rate of fMet–Val forma- terpretation of the general location of the macrolide- tion by fivefold, whereas the rate of formation of binding site, the details of drug placement in ribo- fMet–Phe is reduced by 1,000-fold.34 It is plausible somes of different species vary. In some instances, that even a slight shift in the placement of the C5 these variations are authentic and likely reflect dif- side chain of these antibiotics in ribosomes of differential binding of the antibiotic to different ri- ferent species would differentially affect amino acid bosomes. For example, the varying placement of specificity of their inhibitory action. the telithromycin side chain observed in the crys- Besides the interference with peptide elongation tallized complexes is strongly supported by bio- and inhibition of peptide bond formation (in the chemical data (see below). In the other cases, the case of 16-member-ring macrolides), macrolides are reported discrepancies may stem from variations also shown to promote read through of stop codons in the interpretation of similar structures and re- located close to the translation initiation site, sug- flect a gradual progress to a more accurate fit- gesting their negative effect on the accuracy of trans- ting of the atomic coordinates into the experimen- lation.40 Although this observation is intriguing, it tal electron densities, rather than true structural remains unclear how the interplay of the drug, the variations. The field would significantly benefit if nascent peptide and the ribosome contributes to this the previously determined structures and experi- effect and whether this mode of macrolide action is mental data were reexamined by the original au- manifested only at the early stages of translation. thors in order to clarify the situation. In the ab- Due to the protein-specific mode of macrolide ac- sence of such analyses, we treat all the reported tion, treatment of bacteria with low concentrations structural differences as authentic, even though the of drugs leads to differential inhibition of produc- accuracy of some reported structures have been tion of many cellular polypeptides, including ribo- questioned.39 When discussing the structures, we somal proteins. Unbalanced synthesis of ribosomal will use the following convention for addressing components results in aberrant ribosome assem- , various drug–target complexes: D50S, D. radiodu- bly.41 42 This effect may exacerbate the inhibitory rans large ribosomal subunit (used by the Yonath action of the drugs on translation and cell growth.

38 Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. Kannan & Mankin Species-specific binding and action of macrolides laboratory at the Weizmann Institute, Rehovot, Is- tone ring observed in the ribosomes of other species. rael); H50S, H. marismortui large ribosomal subunit Therefore, it remains a possibility that the existing and T70S, T. thermophilus ribosome (both used for discrepancies in the D50S erythromycin model re- antibiotic studies by the Steitz laboratory at the Yale flect a difference in the interpretation of experimen- University); and E70S, E. coli ribosome (used by the tal data rather than a genuine change in the mode Cate laboratory at the University of California at of binding.45 The angle at which the cladinose and Berkley). desosamine side chains project from the central ring also differs in the D. radiodurans model compared The D. radiodurans 50S subunit compared to the other published structures (Fig. 3). to other ribosomes Varying placement of the alkyl–aryl side The first macrolide–ribosome complexes were re- chain of ketolides ported for the D. radiodurans large ribosomal subunit.18 In the structure of erythromycin bound to One of the distinguishing features of ketolides is D50S, the macrolactone ring was modeled in a the presence of an extended alkyl–aryl side chain high-energy, folded-in conformation, as opposed to that enhances drug binding due to additional in- the low-energy, folded-out conformation reported teractions with the ribosome. This conclusion came later for the drug complexed to H50S, T70S, and from foot-printing studies, carried out with E. coli E70S.19–21,39 The folded-out conformation of the and S. aureus ribosomes, where the side chain of macrolactone is also characteristic of the free drug, ketolides protected A752 in the loop of helix 35 of both in solution and in the crystalline state.43,44 In the 23S rRNA from modification by dimethyl sulfate the H50S, T70S, and E70S structures, macrolactone (DMS).7,8 Several mutations in or near the loop of lays flat against the tunnel wall, whereas in the D. helix 35 conferred resistance to ketolides.8,46 How- radiodurans model, the macrolactone ring of ery- ever, when the first crystallographic structures of thromycin peels off the tunnel wall and projects telithromycin complexed with the D. radiodurans more into the tunnel lumen (Fig. 3). The place- and H. marismortui large ribosomal subunits were ment of the macrolactone of other macrolides, for published,39,47 they failed to support the results of example telithromycin, in D. radiodurans deviates chemical probing or mutational analyses because less from the more common pose of the macrolac- the alkyl–aryl side chain did not approach the loop of helix 35 close enough to explain the biochemical and genetic data (Fig. 4B and C). Furthermore, the orientation of the side chain was drastically different in these two structures. In D50S, the alkyl–aryl side chain of telithromycin stretched down the tunnel and interacted with residue 790 in domain II of 23S rRNA. In contrast, in the H50S complex, the side chain was folded over the macrolactone ring and stacked upon pyrimidine at position 2609 in domain V. Notably, the difference in the placement of the telithromycin alkyl–aryl side chain in the D50S and H50S structures and the deviation from its expected orientation did not appear to be a crystallization artifact. DMS probing experiments carried out with D50S and H50S confirmed the lack Figure 3. The difference in the placement of the erythromycin of telithromycin-dependent protections in the loop macrolactone ring in the D. radiodurans (green, PDB accession of helix 35 in these organisms and further validated number 1JZY)18 and E. coli (salmon, 3OFR)21 ribosome. Des- interactions reported for the “folded over” confor- osamine and cladinose sugars assume a similar position, but mation of the side chain seen in the H50S structure21 the orientation and conformation of the lactone ring is sub- (Xiong and Mankin, unpublished). stantially different. The structure of erythromycin in the E. coli complex is similar to that seen in H. marismortui (1YI2)39 and In view of these findings, the placement of the ke- T. thermophilus (3OHJ)20 ribosomes. tolide alkyl–aryl side chain in the E. coli ribosome,

Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. 39 Species-speciﬁc binding and action of macrolides Kannan & Mankin

Figure 4. Varying placement of the alkyl–aryl side chain of ketolides in ribosomes of different species. (A) In E. coli (PDB accession number 3OAT),21 as well as in T. thermophilus (3OI3 not shown),20 the alkyl–aryl side chain of telithromycin (indicated by a black triangle) stacks upon the A752-U2609 base pair. (B) In D. radiodurans (1P9X)47 the side chain extends down the tunnel but does not come close to position 752. (C) In H. marismortui (1YIJ)39 the side chain folds over the macrolactone ring. The positions of the desosamine sugar (indicated by open triangles) remain fairly invariant in different structures. or for that matter in ribosomes of Gram-positive coli ribosomes, all the ketolides afford the same pathogens, remained a mystery until 2010 when the strong protection of A752 from DMS modifica- J. Cate and T. Steitz laboratories reported the high- tion, indicating that the placement of the side chain resolution structures of telithromycin complexed to should be fairly invariant. However, in D50S com- 70S ribosomes from E. coli and T. thermophilus.20,21 plexed with cethromycin, a ketolide structurally In these new structures, the aromatic head of the similar to telithromycin but carrying a distinct telithromycin side chain closely approached the loop alkyl–aryl side chain at the C6 position rather than of helix 35 in domain II of 23S rRNA and established at C11 as in telithromycin, the placement of the stacking interactions with the base pair formed be- side chain notably deviates from that reported for tween A752 in this loop and U2609 in domain V the telithromycin-D50S complex.47,48 In the D. ra- (Fig. 4A). This interaction was in excellent agree- diodurans and H. marismortui ribosomes that lack ment with the reported protection of A752 in the E. the A752–U2609 docking platform, the alkyl–aryl coli ribosome from DMS modification. Cate et al. chain apparently fails to lock in its proper place and noted that formation of the A752–U2609 base pair adopts varying conformations that are likely irrele- is impossible in H. marismortui where U2609 of vant to the placement of the drug in the ribosomes the E. coli 23S rRNA is replaced with C, or in D. of Gram-positive pathogens. radiodurans, where C substitutes for A752.21 Im- The second azithromycin site in portantly, the nucleotide sequences of 23S rRNA D. radiodurans of many Gram-positive pathogens, including S. aureus and S. pneumoniae arecompatiblewiththe It is commonly assumed that most antibiotics have formation of the A752–U2609 base pair, indicat- been evolutionarily selected to act upon one spe- ing that the placement of the ketolide’s alkyl–aryl cific target site. Surprisingly, crystallographic stud- side chain observed in the E70S and T70S struc- ies have shown that some protein synthesis in- tures likely resembles its position in the ribosomes hibitors can bind to several sites in the ribosome. of clinical strains. Yet the exact position of the side For example, aminoglycosides bind to helix 44 in chain may slightly deviate in pathogens because the 16S rRNA as well as helix 69 in 23S rRNA of the E. A752 protection observed in S. aureus was some- coli ribosome,49 whereas up to five binding sites have what less pronounced than that observed in E. coli been reported for tetracycline in the small subunit of ribosomes.21 the T. thermophilus ribosome.50,51 The existence of The structure and the site of attachment of the secondary antibiotic sites may simply stem from the alkyl–aryl side chain to the macrolactone ring vary large size of the ribosome, which offers many cavi- among different ketolides. Nevertheless, in the E. ties that provide a favorable geometry and chemical

40 Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. Kannan & Mankin Species-speciﬁc binding and action of macrolides

away from the PTC, where it is inserted at the tunnel constriction between the extensions of proteins L4 and L22. The distinct amino acid sequences of the L4 and L22 proteins could potentially account for the binding of the second azithromycin molecule in the D. radiodurans ribosome. In the second site, the drug interacts with the amino acid residues Tyr59, Gly60,Gly63, and Thr64 of L4 (D. radiodurans numbering is used for the D50S ribosomal proteins). Of these, Gly60 is the least conserved and is frequently replaced by Lys or Arg (or more rarely Pro or Ala). The presence of the bulkier amino acids at this L4 position in the ribosomes of most bacteria could interfere with binding of azithromycin to the second site. Similarly, Arg111 of the L22 protein, with which azithromycin interacts in its D50S second-binding site, is not well conserved and, thus, may account for the unusual drug binding properties of the D. radiodurans ribosome. When bound in the neighboring sites in D50S, Figure 5. Two azithromycin binding sites in the D.radiodurans the two azithromycin molecules come close enough ribosome.48 The first molecule of azithromycin (Azm I) binds to each other to establish a direct hydrogen bond in the conventional macrolide binding site; the second molecule interaction between the N2 of the desosamine of (Azm II) binds farther down the tunnel. The loops of proteins the drug in the second site and O12 of the lactone L4 and L22 that form the tunnel constriction and contribute to ring of azithromycin in the canonical binding site theAzmIIbindingareshowninblue.Ahydrogenbondbetween Azm I and Azm II is shown by a dotted line. (Fig. 5). Hypothetically, such an interaction can lead to binding cooperativity, which could enhance the affinity of the drug for the ribosome. A similar bind- environment for drug binding;52,53 binding of a ing of two drug molecules to D50S was also reported drug in such sites is not expected to produce any for bridged ketolides.55 While foot-printing studies specific effect on translation. On the other hand, it failed to support the existence of a second-macrolide is conceivable that some antibiotics have been evo- molecule in the D50S,56 drug-binding experiments lutionarily optimized for interacting with more than have shown that the D. radiodurans ribosome can one ribosomal site in order to achieve the full extent bind two macrolide molecules in solution.54 If, the of their inhibitory action. binding of two macrolides in the D50S NPET is Biochemical and crystallographic analyses of in- confirmed, it could open an interesting avenue for teractions of macrolides with the E. coli ribosome, optimizing macrolide drugs to stimulate their bind- as well as with ribosomes of H. marismortui and ing to neighboring sites in the tunnel of ribosomes T. thermophilus, consistently prove the existence of bacterial pathogens. of a unique macrolide binding site located in the Unusual binding of troleandomycin in the NPET near the PTC.19,39,54 Surprisingly, however, D. radiodurans large ribosomal subunit the binding of two azithromycin molecules was observed in the tunnel of the D. radiodurans large ribo- Troleandomycin (triacetyloleandomycin) is a 14- somal subunit48 (Fig. 5). In D50S, one azithromycin member-ring macrolide derived by the acetylation molecule is found at the canonical macrolide site of its natural precursor oleandomycin, at three sites: where the central macrolactone ring of the drug, at the C12 position of the lactone ring, the 2 position the desosamine and cladinose side chains assume a of C3 oleandrose sugar, and the 4 of the C5 amino position similar to that seen for macrolides in other sugar (Fig. 1A). In the crystallographic structure species. The second azithromycin molecule binds of troleandomycin complexed with D50S, the drug in the D50S NPET next to the first one, but farther binds in the NPET, however at a site significantly

Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. 41 Species-speciﬁc binding and action of macrolides Kannan & Mankin

One of the most frequently found mutations that renders cells highly resistant to most 14- and 15- member ring macrolides, including ketolides, is an adenine to guanine transition at position 2058 in 23S rRNA (see Vester et. al.59 for review). However, in S. pneumoniae, the A2058G mutation has little effect on susceptibility to ketolides.46,60 The reason for the unusual behavior of the S. pneumoniae A2058G mutant was traced to the nature of the neighboring 2057–2611 base pair that forms a part of the cradle hosting the macrolactone ring in the ribosome (see Fig. 2C).61 In many bacteria, including M. smegmatis, the position 2057 is occupied by an adenine paired to uridine at position 2611. In this back- Figure 6. Different binding modes of troleandomycin in the ground, the A2058G mutation confers resistance to large ribosomal subunits of D. radiodurans and H. marismortui. In the H.marismortui ribosome(PDBaccessionnumber3I56),58 a high concentration of the ketolide, telithromycin. the placement of troleandomycin (salmon) is similar to that of However, when the 2057A–2611U pair is replaced other macrolides. In D. radiodurans (1OND),57 troleandomycin with G–C, as in S. pneumoniae, the extent of ke- molecule (green) is shifted down the tunnel and makes contacts tolide resistance conferred by the A2058G mutation with the amide nitrogen of Ala2 of ribosomal protein L32 (dotted is significantly diminished. Presumably, when the line). The position of A2058 is shown for reference. antibiotic binds with high affinity to the wild-type (A2058) ribosome, the identity of the 2057–2611 base pair does not play a major discriminatory role. shifted “down” the tunnel relative to the conven- However, when the drug binding is weakened by tional macrolide-binding site (Fig. 6).57 Binding of the A2058G mutation, the nature of the base-paired troleandomycin to D50S was reported to promote residues 2057 and 2611 becomes important. the rearrangement of ␤-hairpin of the L22 protein.57 In addition to the direct effect of species-specific In contrast to the D50S structure, in H50S, trolean- nucleotide differences upon antibiotic binding, domycin binds at the conventional macrolide site variations in ribosome structure may affect the fit- and does not reorient the L22 hairpin.58 In D50S, ness cost of the resistance mutations and therefore, the binding of troleandomycin to an alternative site the prevalence of specific mutations in different or- couldbefavoredduetotheformationofahydro- ganisms. For example, in pneumococci, the A2059G gen bond between the Ala2 amide of the protein L32 resistance mutation is observed far more frequently and the oxygen atoms of the C9 oxirane and the than the A2058G mutation, which is widespread in C10 keto-group of troleandomycin.57 Archeal ribo- majority of other bacterial species.59,62 The A2058C somes do not contain an equivalent protein, whereas mutation occurs frequently in Helicobacter pylori E. coli contains L32, but the protein lacks the first and confers a high level of macrolide resistance; four amino acid residues that are present in the D. however, this mutation is deleterious in E. coli and radiodurans L32 and thus may not support drug thus usually does not show up in selection exper- binding in the alternative site.45 iments.63,64 The A2058U mutation provides sig- nificant macrolide resistance in E. coli but only Species-specific effect of macrolide , moderate resistance in H. pylori.63 65 Although no resistance mutations exhaustive analyses of protein mutations have been Interspecies variations in the ribosome structure af- carried out, different amino acid residues of pro- fect the binding of macrolides not only to wild-type teins L22 and L4 are usually found to be mutated ribosomes, but also to ribosomes that have acquired in macrolide–resistant isolates of varying bacte- resistance mutations. As a result, a mutation that rial species (reviewed by Franceschi et al.62). Such makes one organism resistant to high concentra- species-specific biases in occurrence of resistance tions of an antibiotic may only have a mild effect in mutations show that variations in the ribosome another species. structure may influence both, the fitness cost of

42 Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. Kannan & Mankin Species-specific binding and action of macrolides resistance as well as the differential binding of the drug to the wild-type and mutant ribosomes in different bacteria. The role of the posttranscriptional modifications in species-specific mode of macrolide binding A number of indigenous rRNA modifying enzymes methylate or pseudouridylate-specific rRNA nucleotides at functionally important regions in the ribosome. Although the mission of rRNA modifications remains an enigma, it is generally be- lieved that they fine-tune the functions of the ribosome in translation. Approximately one-third of modified residues of the 23S rRNA are clustered around the NPET and some of them directly affect rRNA segments that constitute the macrolide- binding site. Posttranscriptional modifications that alter the chemical makeup of the binding site may have an important influence upon macrolide bind- Figure 7. Posttranscriptionalmodificationsintheloopofhelix ing. One such heavily modified rRNA segment is 35 of 23S rRNA. Shown is the secondary structure of helix 35 in the loop of helix 35 in 23S rRNA, which in in the E. coli ribosomes where three positions, G745, U746, and 1 5 1 5 U747 are modified to m G, Ψ and m U, respectively. Gram- E. coli includes m G745, 746, and m U747 (see , positive bacteria usually carry m1G748 instead of m1G745.66 67 Refs. 26,66–68)(Fig. 7). The modification pattern In some bacterial species, the loop of helix 35 is unmodified.69 varies significantly between ribosomes of differ- Enzymes responsible for specific modifications are indicated. ent bacterial species and influences antibiotic binding.69 TheenzymeRlmAI responsible for the N1 methylation of G745 is found predominantly in E. coli hypersensitive to several PTC-targeting an- Gram-negative bacteria.26 In Gram-positive organ- tibiotics.72 Therefore, it is expected that organisms isms, RlmAI is replaced with RlmAII, which adds lacking the RluC enzyme responsible for U2504 a methyl group to N1 of the neighboring G748.70 modification would exhibit hypersusceptibility to When A2058 is monomethylated by the action 16-member-ring macrolides that reach into the of an Erm enzyme, the methylation of G748 by PTC active site. RlmAII confers resistance to tylosin.26 Therefore, The variations in the NPET structure affect the A2058 monomethyltransferase would render the mode of action of macrolides Gram-positive bacteria that carry the rlmAII gene resistant to tylosin, but would fail to do so with It has long been thought that macrolides in- Gram-negative organisms that lack rlmA.II hibit translation of all proteins because they The loop of helix 35 in 23S rRNA is close to the “plug” the NPET. However, recent structural ev- NPET constriction formed by proteins L4 and L22. idence shows that blocking of the tunnel by the The macrolide resistance mutations in L22 alter the macrolide molecule is incomplete.39 Some pro- conformation of nucleotides 747 and 748.71 Thus, teins can successfully thread through the tunnel the natural diversity in the modification pattern of narrowed by the antibiotic and evade macrolide this rRNA segment may hypothetically modulate inhibition28,73 (Kannan et al.,inpreparation).In the allosteric effects of the L22 and L4 mutations. the conventional plug scenario, the structure of the Species-specific difference in the post- tunnel would have little relevance to the mode of transcriptional modification pattern in other macrolide action. However, the ability of the nascent sites of 23S rRNA may also affect the binding of polypeptide to slither through the narrowed crawl macrolide antibiotics. For instance, the lack of space in the drug-obstructed NPET should be crit- pseudouridylation of U2504 was shown to render ically influenced by the tunnel architecture, which

Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c 2011 New York Academy of Sciences. 43 Species-speciﬁc binding and action of macrolides Kannan & Mankin

Figure 8. The variation in the ribosome tunnel structure between bacterial species. (A) The 23S rRNA residues located in the NPET and differing between Gram-negative E. coli and Gram-positive B. subtilis. Segments of 23S rRNA that form the walls of the exit tunnel are indicated by thick lines on the 23S rRNA secondary structure diagram and nucleotide variations are indicated by red arrowheads. (B) Difference in the shape of the exit tunnels in ribosomes of Gram-negative (E. coli, green) and Gram-positive (D. radiodurans, orange) bacteria. The figure is reproduced with permission from the reference.74 shows substantial variation between different bac- ribosomes of pathogenic strains. The first efforts in teria74 (Fig. 8). this direction illustrate the promise of structure- We have recently discovered that bacterial cells based drug design.75 Expanding the number of treated with very high (100-fold MIC) concentra- species from which crystallizable ribosomes can be tions of erythromycin or telithromycin can still effi- obtained, while ensuring that new structures remain ciently synthesize a subset of polypeptides (Kannan in the public domain, could significantly accelerate et al., in preparation). Importantly, the spectrum of development of new, useful antibiotics and/or stim- such “escape” proteins vary substantially between ulate novel applications of known drugs. Gram-negative E. coli and Gram-positive S. aureus likely reflecting different architecture of the NPETs Acknowledgments in the ribosomes of these organisms. The nature of We thank Lisa K. Smith and Jacqueline M. LaMarre proteins that escape macrolide inhibition are likely for proofreading the manuscript and Artem Mel- to influence cell growth and viability. Therefore, the man for verifying the antibiotic structures. The work variation in the structure of the ribosomal tunnel related to the sites of antibiotic action in this lab- in the vicinity of the macrolide binding site could oratory is supported by a grant from the United be a major contributing factor to the differential ef- States-Israel Binational Science Foundation (No. fect of specific macrolide antibiotics upon different 2007453). bacterial species. Conflicts of interest Concluding remarks The authors declare no conflicts of interest. Here, we presented several examples illustrating how macrolide binding and action, as well as bac- References terial resistance to these antibiotics, can be affected 1. McGuire, J.M., R.L. Bunch, R.C. Anderson, et al. 1952. Ilo- by the species-specific traits of ribosome structure. tycin, a new antibiotic. Schweiz Med. Wochenschr. 82: 1064– There is no doubt that the use of model organ- 1065. isms was incredibly important for understanding 2. Ban, N., P. Nissen, J. Hansen, et al. 2000. The complete the basic principles of binding and action of these atomic structure of the large ribosomal subunit at 2.4 A˚ drugs. However, structure-guided efforts for the de- resolution. Science 289: 905–920. 3. Vazquez, D. 1977. Inhibitors of Protein Synthesis. Springer. velopment of novel antibiotics need to take into ac- New York. count the interspecies variations in ribosome struc- 4. Poulsen, S.M., C. Kofoed & B. Vester. 2000. Inhibition of ture and possible differences in drug binding sites in the ribosomal peptidyl transferase reaction by the mycarose

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