Macrolide antibiotics in the ribosome exit tunnel: species … · Kannan & Mankin Species-speciﬁc...

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

ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Antimicrobial Therapeutics Reviews

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

Krishna Kannan and Alexander S. MankinCenter 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 atChicago, 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. Themajority of information on the principles of binding and action of these antibiotics comes from studies that employedmodel organisms. However, there is a growing understanding that the binding of macrolides to their target, as well asthe mode of inhibition of translation, can be strongly influenced by variations in ribosome structure between bacterialspecies. Awareness of the existence of species-specific differences in drug action and appreciation of the extent ofthese differences can stimulate future work on developing better macrolide drugs. In this review, representative casesillustrating the organism-specific binding and action of macrolide antibiotics, as well as species-specific mechanismsof resistance are analyzed.

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

Introduction

Macrolides are an important class of antibiotics ef-fective against a number of pathogenic bacteria.These mostly bacteriostatic drugs are commonlyused to treat respiratory tract infections includ-ing community acquired pneumonia, pharyngitis,and tonsillitis, along with skin and soft tissue in-fections, urogenital infections, and orodental in-fections. Macrolides were introduced into medi-cal practice almost 60 years ago and continue tobe viewed as excellent antibiotics with high po-tency and low toxicity. Erythromycin A, a nat-ural product of Saccharopolyspora erythraea, wasthe first macrolide to be advanced to medical usein the early 1950s for the treatment of bacte-rial infections.1 Erythromycin (Fig. 1A) is effectiveagainst many Gram-positive pathogenic bacteria in-cluding Staphylococcus aureus, Streptococcus pneu-moniae, S. pyogenes, and Enterococcus sp.; someGram-negative bacteria such as Neisseria gonor-rhoeae, Bordetella pertussis, and Haemophilus in-fluenzae; and intracellular pathogens such as My-coplasma sp., Legionella sp., and Chlamydia sp.Semisynthetic derivatives of erythromycin, includ-

ing drugs such as clarithromycin, roxithromycin,and azithromycin (Fig. 1A and B), which be-long to the second generation of macrolides, ex-hibit increased acid stability, better oral bioavail-ability, improved pharmacodynamics, and broaderantimicrobial spectrum. The second-generationmacrolides showed particularly improved activ-ity against Haemophilus influenzae and intracel-lular pathogens such as Mycobacterium avium-intracellulare. The emergence and broad spreadof resistance prompted the development of thenew generation of macrolides. Ketolides (Fig. 1C),representing the third generation, show improvedpotency against many sensitive and some resis-tant strains and are often associated with bacte-ricidal activity. Macrolide antibiotics with an ex-tended macrolactone ring, such as 16-memberedmacrolides (Fig. 1D), find extensive use in veterinarymedicine and are sometimes also used in humans.

Macrolides inhibit bacterial growth by binding tothe ribosome and blocking protein synthesis. The ri-bosome is an RNA-based macromolecular machinewith a molecular weight of ca. 2.5 million Da (in bac-teria). It represents one of the best cellular antibiotictargets. The ribosome is composed of two subunits,

doi: 10.1111/j.1749-6632.2011.06315.xAnn. 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 onthe erythromycin structure. Side chains discussed in the review are labeled.

small and large (also known as the 30S and 50Ssubunits, respectively, based on their sedimentationcoefficients). The decoding center in the small ribo-somal subunit is responsible for the selection of theaminoacyl-tRNAs in based on the order of codonsin mRNA. The amino acids are assembled into apolypeptide chain in the peptidyl transferase center(PTC) located in the large ribosomal subunit. Thenewly assembled polypeptides leave the ribosomethrough the nascent peptide exit tunnel (NPET),which starts at the PTC and spans the body of thelarge subunit2 (Fig. 2A and 2B). Macrolides bind inthe NPET close to the PTC. They hinder the passageof the newly synthesized polypeptides through thetunnel, thereby interrupting translation elongation,most commonly at the early rounds of protein syn-thesis.3 In addition, some macrolides with extendedside chains reach close to the catalytic center andinterfere with peptide bond formation.4

Most of what is known about the interactionsof protein synthesis inhibitors with their targetscomes from the studies of ribosomes from selectedmodel organisms. Powerful biochemical techniqueshave been used to locate drug binding sites in Es-cherichia coli ribosomes.5–8 Additional informationabout drug–ribosome interactions came from ge-netic studies that were employed to identify re-sistance mutations in E. coli ribosomal proteinsand rRNA.9–14 These studies were later expandedand enhanced by including organisms with sim-pler rRNA genetics, such as the halophilic archeonHalobacterium halobium15,16 and engineered strainsof Mycobacterium smegmatis,17 which carry sin-gle rDNA alleles. Subsequent advent of ribosomecrystallography resulted in atomic-level models ofribosome-drug complexes.18–21 However, since onlya few types of ribosomes are amenable to crystalliza-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 obstructthe exit tunnel. View down NPET from the interface side of the large ribosomal subunit. The erythromycin molecule is shown insalmon. (B) Erythromycin binds in the exit tunnel close to the PTC. Small (30S) and large (50S) ribosomal subunits are coloredyellow 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 sitein 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 A752and G748 and ribosomal proteins L4 and L22 (H. marismortui 50S subunit, PDB accession number 1K9M).19

by using the ribosomes and ribosomal subunitsfrom a very limited set of species—the halophilicarcheon Haloarcula marismortui, the radiation-resistant Gram-positive Deinococcus radiodurans,the Gram-negative mesophilic E. coli, and the ther-mophilic Thermus thermophilus.

Most of the aforementioned experimental mod-els used in biochemical, genetic, or crystallographicstudies are only distantly related to clinically relevantpathogens. Because the ribosome structure is highlyconserved, results obtained with model organismsare often directly and sometimes indiscriminately

Ann. N.Y. Acad. Sci. 1241 (2011) 33–47 c© 2011 New York Academy of Sciences. 35


extrapolated to pathogenic bacterial species. How-ever, in many instances, the species-specific idiosyn-crasies in the ribosome structure result in importantdifferences in drug binding and action.

In this short review, we will first provide a generaloverview of the mode of macrolide binding to theribosome and the current view of the mechanism ofdrug action. We will then discuss several examplesdemonstrating that the binding of macrolides, andthus the mode of their action, can vary substantiallyin different species.

Chemical structure of macrolide antibiotics

Clinically useful macrolides are characterized by thepresence of a 14-, 15-, or 16-atom macrolactonering, to which several neutral or amino sugars andother side chains are attached (Fig. 1). The proto-typical natural macrolide antibiotic, erythromycin,is composed of a 14-member lactone ring that car-ries two sugars, cladinose, and desosamine, attachedat positions C3 and C5, respectively (Fig. 1A). In thesemisynthetic azithromycin (Fig. 1B), the macrolac-tone ring of erythromycin is extended by an addi-tional nitrogen atom. The 16-membered macrolideslike tylosin or carbomycin contain an extendeddisaccharide at the C5 position and often possessseveral additional side chains attached at other po-sitions of the macrolactone (Fig. 1D). In the third-generation macrolides, the ketolides, a keto groupreplaces the C3 cladinose of erythromycin (Fig. 1C).In addition, ketolides carry a 11,12 cyclic carbamateand an extended alkyl–aryl side chain that can belinked at different sites to the macrolactone moiety.Some clinically promising ketolides are additionallyfluorinated at C2.22,23

Both the macrolactone ring and the side chainsof macrolide antibiotics contribute to the bindingaffinity of the drug to the ribosomal target. How-ever, while variation in the structure of the centralmacrolactone ring has little influence on the modeof drug binding or inhibition of translation,19,21 thestructure of the side chains directly affects the inter-action of the drug with specific rRNA residues, themechanism of macrolide action, and its propensityto activate resistance mechanisms.

Macrolide-binding site in the ribosome

Macrolides bind in the upper chamber of the NPET,between the PTC and the constriction formed byproteins L4 and L22 (Fig. 2B). The binding site is

composed predominantly of rRNA nucleotides be-longing to domains II and V of the 23S rRNA. Thecentral macrolactone ring of the drug establisheshydrophobic interactions with the rRNA residues2057, 2611, and 2058 (E. coli numbering of rRNAnucleotides is used throughout) that form the tun-nel wall on the side of the PTC A site. In most ofthe available high-resolution crystallographic struc-tures, the macrolactone is positioned flat against thewall with the side chains protruding either up thetunnel, toward the PTC active site, or down, to-ward the tunnel constriction. The C5 amino sugar(desosamine in 14- and 15-member ring macrolidesor mycaminose of the mycarose-mycaminose disac-charide in the 16-member ring macrolides) extendsin the direction of the PTC and is drawn close to thecrevice between the bases of the adenine residuesA2058 and A2059. A hydrogen bond donated bythe 2′-OH of the desosamine to the N1 of A2058additionally contributes to drug binding. The inter-actions of the C5 side chain with A2058 and A2059are very important: dimethylation of the exocyclicamine of A2058 by erythromycin resistance methyl-transferases (Erms) or mutations of A2058 or A2059dramatically reduce the affinity of all macrolides forthe ribosome.14,24 While the C5 desosamine of the14- and 15-member ring macrolides does not reachthe PTC, the longer disaccharide C5 side chains of16-member ring macrolides approaches the PTCactive site more closely and can affect the cataly-sis of peptide bond formation. The C5 disaccharidein carbomycin and josamycin is further extended byan isovalerate moiety (Figs. 1D and 2D) that reachesdirectly into the PTC A site, where it is positionedin the hydrophobic crevice formed by residuesA2451 and C2452 in the heart of the catalyticcenter.4,19

The cladinose sugar attached at C3 of ery-thromycin and second-generation macrolidescomes into close contact with C2610 and G2505(Fig. 2C).19,21 Although the C2610 mutation has lit-tle effect upon drug binding or susceptibility, thisnucleotide is apparently important for fine-tuninginteractions among the drug, the ribosome, andthe nascent peptide because the C2610U muta-tion affects the ability of the cladinose-containingmacrolides to activate the expression of drug-inducible erm genes.25

The C3 cladinose is absent in ketolides and 16-member ring macrolides. However, the presence of



an extended alkyl–aryl side chain in ketolides or ad-ditional side chains in 16-member ring macrolidescompensate for the absence of cladinose, becausethey increase the drug’s affinity by establishing ad-ditional interactions with nucleotides of domain IIof the 23S rRNA. Recent crystallographic structuresof telithromycin, the first clinically approved ke-tolide, complexed to the E. coli or T. thermophilusribosomes confirmed the previously proposed closecontacts of the side chain with the loop of helix35.20,21 Similarly, the C14-linked mycinose of tylosininteracts with bases at positions 748, 751, and 752 inthe helix 35 loop19 (Fig. 2E). The ribosomal contactsof mycinose are clearly important for tylosin bind-ing, because when these interactions are abolishedby the methylation of A748 by RlmAII methyltrans-ferase (in conjunction with the A2058 monomethy-lation), tylosin fails to inhibit cell growth.26

Some 16-membered macrolides (e.g., tylosin)carry an ethyl-aldehyde group at the C6 position.In the crystallographic structures of these drugscomplexed with the H. marismortui 50S subunit, acontinuous electron density connects the aldehydegroup to the exocyclic amine of A2062, which iscompatible with the formation of a reversible cova-lent bond.19 A similar covalent bond was observedbetween josamycin and A2062 in the D. radiodu-rans 50S subunit.27 Abolishing the formation ofthis putative bond is associated with decreased drugaffinity and reduced ability of the drug to inhibittranslation.28

Some of the nucleotides that form the macrolide-binding site, for example, the 2057–2611 base pair orthe loop of helix 35, exhibit considerable variationamong bacterial species. However, even the place-ment of conserved rRNA residues involved in drugbinding may vary in different ribosomes becauseof the species-specific interactions with the non-conserved second-shell nucleotides that buttress thedrug binding site components. Therefore, it is notso surprising that the exact orientation of the samemacrolide molecule bound to ribosomes of differentspecies can exhibit substantial variation (see below).

Although most of the interactions that macrolidesestablish with the ribosome involve rRNA, long sidechains of some 16-member ring macrolides and theketolides extend far enough down the tunnel tocome into direct contact with the ribosomal pro-teins L4 and L22, which form the constriction ofthe NPET ca. 20–35 A away from the PTC.2 For ex-

ample, the C14-linked mycinose of tylosin directlyinteracts with protein L22 (Fig. 2E), whereas theC9-linked forosamine of spiramycin reaches closeto L4.19 Mutations in these proteins confer resis-tance to various macrolides. However, the effects ofribosomal protein mutations upon binding of mostmacrolide antibiotics could be either allosteric orinvoke unconventional mechanisms.29,30 The struc-ture of the ribosomal proteins is less conserved thanthat of the rRNA. Variation in protein structuremay contribute to the ribosome-specific binding ofmacrolides.

Mechanism of action

According to the commonly accepted model,macrolide antibiotics inhibit protein synthesis byobstructing the growth of the nascent peptide chain.The 14- and 15-atom lactone ring macrolides havelittle effect during the early rounds of translation.Only after the first few amino acids are polymer-ized and the growing polypeptide reaches the siteof drug binding does the subsequent progressionof the peptide through the NPET become inhib-ited, and peptidyl-tRNA dissociates from the ribo-some.31,32 With the cladinose containing 14- and15-membered macrolides bound in the tunnel, thedissociated peptidyl-tRNAs carry a 6–9 amino acid–long peptide.33 Ketolides, which lack the C3 cladi-nose, allow polymerization of 9–10 amino acids.33

Accumulation of peptidyl-tRNA, leading to the ex-haustion of the pools of free tRNAs in the cell,could be the major cause of translation cessationin macrolide-treated cells.32

When the drug obstructs the growth of thenascent peptide, the peptidyl-tRNA drop-off rateand the spontaneous dissociation rate of the drugfrom the ribosome have important consequences.34

If the antibiotic dissociates prior to the peptidyl-tRNA drop-off and a few amino acids are added tothe polypeptide before a new antibiotic moleculebinds, then the synthesis of the protein will becarried to its completion because longer nascentpolypeptides prevent drug rebinding.35 Because ofthat, some residual protein synthesis can be ob-served even when cells are treated with a highconcentration of drugs. The dissociation rate ofpeptidyl-tRNA depends on the nascent peptidesequence, length, and its interaction with the NPET,whereas the dissociation rate of the drug is deter-mined by its structure and the specifics of its contacts



with the NPET in the drug binding site.36 There-fore, the residual protein synthesis afforded duringmacrolide treatment can be protein specific,28 andthus, species specific. Furthermore, some nascentpeptides have the capacity to evict the drug from theribosome,37,38 whereas others can thread throughthe tunnel obstructed by the macrolide antibiotic39

(Kannan et al., in preparation). These aspects maycontribute to the idiosyncratic action of macrolideantibiotics against different bacterial species.

In contrast to 14- and 15-member ring macrolidesthat do not inhibit polymerization of the first fewamino acids, the 16-membered macrolides thatcarry a disaccharide (mycaminose–mycarose) sidechain at the C5 position can block translation at ear-lier stages. The mycaminose–mycarose chain thatreaches into the PTC can directly interfere withthe formation of the second or even first peptidebonds.4 In carbomycin A and josamycin, the disac-charide molecule is further extended by isovalerateappended to the mycarose sugar. This group reachesinto the amino acid binding pocket of the PTC A-site. Therefore, these drugs can affect peptide bondformation in an amino acid-specific manner. Thus,josamycin diminishes the rate of fMet–Val forma-tion by fivefold, whereas the rate of formation offMet–Phe is reduced by 1,000-fold.34 It is plausiblethat even a slight shift in the placement of the C5side chain of these antibiotics in ribosomes of dif-ferent species would differentially affect amino acidspecificity of their inhibitory action.

Besides the interference with peptide elongationand inhibition of peptide bond formation (in thecase of 16-member-ring macrolides), macrolides arealso shown to promote read through of stop codonslocated close to the translation initiation site, sug-gesting their negative effect on the accuracy of trans-lation.40 Although this observation is intriguing, itremains unclear how the interplay of the drug, thenascent peptide and the ribosome contributes to thiseffect and whether this mode of macrolide action ismanifested only at the early stages of translation.

Due to the protein-specific mode of macrolide ac-tion, treatment of bacteria with low concentrationsof drugs leads to differential inhibition of produc-tion of many cellular polypeptides, including ribo-somal proteins. Unbalanced synthesis of ribosomalcomponents results in aberrant ribosome assem-bly.41,42 This effect may exacerbate the inhibitoryaction of the drugs on translation and cell growth.

Species-specific macrolide–ribosomeinteractions

The resolution of the currently available structuresof ribosome–macrolide complexes range from 2.5 Ato 3.5 A, which makes it possible to examine drug–target interactions at an atomic level. However, itis important to remember that the X-ray structuresneed to be treated with a certain degree of discre-tion. While the binding and the action of macrolideantibiotics are likely to be influenced by the nascentpeptide, none of the structures solved to date containpeptidyl–tRNA. Therefore, these structures reflectthe initial binding mode of the antibiotic to the va-cant ribosome, rather than when the drug elicits itsinhibitory action on translation. Furthermore, crys-tallization conditions significantly deviate from theenvironment in which the ribosome exists in the cellcytoplasm, which may influence the conformationof the ribosome, its dynamics, and its interactionswith antibiotics. In addition, as mentioned earlier,none of the published structures employ ribosomesfrom organisms closely related to pathogenic Gram-positive bacteria.

Although the available structures converge in in-terpretation of the general location of the macrolide-binding site, the details of drug placement in ribo-somes of different species vary. In some instances,these variations are authentic and likely reflect dif-ferential binding of the antibiotic to different ri-bosomes. For example, the varying placement ofthe telithromycin side chain observed in the crys-tallized complexes is strongly supported by bio-chemical data (see below). In the other cases, thereported discrepancies may stem from variationsin the interpretation of similar structures and re-flect a gradual progress to a more accurate fit-ting of the atomic coordinates into the experimen-tal electron densities, rather than true structuralvariations. The field would significantly benefit ifthe previously determined structures and experi-mental data were reexamined by the original au-thors in order to clarify the situation. In the ab-sence of such analyses, we treat all the reportedstructural differences as authentic, even though theaccuracy of some reported structures have beenquestioned.39 When discussing the structures, wewill use the following convention for addressingvarious drug–target complexes: D50S, D. radiodu-rans large ribosomal subunit (used by the Yonath



laboratory at the Weizmann Institute, Rehovot, Is-rael); H50S, H. marismortui large ribosomal subunitand T70S, T. thermophilus ribosome (both used forantibiotic studies by the Steitz laboratory at the YaleUniversity); and E70S, E. coli ribosome (used by theCate laboratory at the University of California atBerkley).

The D. radiodurans 50S subunit comparedto other ribosomes

The first macrolide–ribosome complexes were re-ported for the D. radiodurans large ribosomal sub-unit.18 In the structure of erythromycin bound toD50S, the macrolactone ring was modeled in ahigh-energy, folded-in conformation, as opposed tothe low-energy, folded-out conformation reportedlater for the drug complexed to H50S, T70S, andE70S.19–21,39 The folded-out conformation of themacrolactone is also characteristic of the free drug,both in solution and in the crystalline state.43,44 Inthe H50S, T70S, and E70S structures, macrolactonelays flat against the tunnel wall, whereas in the D.radiodurans model, the macrolactone ring of ery-thromycin peels off the tunnel wall and projectsmore into the tunnel lumen (Fig. 3). The place-ment of the macrolactone of other macrolides, forexample telithromycin, in D. radiodurans deviatesless from the more common pose of the macrolac-

Figure 3. The difference in the placement of the erythromycinmacrolactone ring in the D. radiodurans (green, PDB accessionnumber 1JZY)18 and E. coli (salmon, 3OFR)21 ribosome. Des-osamine and cladinose sugars assume a similar position, butthe orientation and conformation of the lactone ring is sub-stantially different. The structure of erythromycin in the E. colicomplex is similar to that seen in H. marismortui (1YI2)39 andT. thermophilus (3OHJ)20 ribosomes.

tone ring observed in the ribosomes of other species.Therefore, it remains a possibility that the existingdiscrepancies in the D50S erythromycin model re-flect a difference in the interpretation of experimen-tal data rather than a genuine change in the modeof binding.45 The angle at which the cladinose anddesosamine side chains project from the central ringalso differs in the D. radiodurans model comparedto the other published structures (Fig. 3).

Varying placement of the alkyl–aryl sidechain of ketolides

One of the distinguishing features of ketolides isthe presence of an extended alkyl–aryl side chainthat enhances drug binding due to additional in-teractions with the ribosome. This conclusion camefrom foot-printing studies, carried out with E. coliand S. aureus ribosomes, where the side chain ofketolides protected A752 in the loop of helix 35 ofthe 23S rRNA from modification by dimethyl sulfate(DMS).7,8 Several mutations in or near the loop ofhelix 35 conferred resistance to ketolides.8,46 How-ever, when the first crystallographic structures oftelithromycin complexed with the D. radioduransand H. marismortui large ribosomal subunits werepublished,39,47 they failed to support the results ofchemical probing or mutational analyses becausethe alkyl–aryl side chain did not approach the loopof helix 35 close enough to explain the biochemicaland genetic data (Fig. 4B and C). Furthermore, theorientation of the side chain was drastically differ-ent in these two structures. In D50S, the alkyl–arylside chain of telithromycin stretched down the tun-nel and interacted with residue 790 in domain IIof 23S rRNA. In contrast, in the H50S complex,the side chain was folded over the macrolactonering and stacked upon pyrimidine at position 2609in domain V. Notably, the difference in the place-ment of the telithromycin alkyl–aryl side chain inthe D50S and H50S structures and the deviationfrom its expected orientation did not appear to bea crystallization artifact. DMS probing experimentscarried out with D50S and H50S confirmed the lackof telithromycin-dependent protections in the loopof helix 35 in these organisms and further validatedinteractions reported for the “folded over” confor-mation of the side chain seen in the H50S structure21

(Xiong and Mankin, unpublished).In view of these findings, the placement of the ke-

tolide alkyl–aryl side chain in the E. coli ribosome,



Figure 4. Varying placement of the alkyl–aryl side chain of ketolides in ribosomes of different species. (A) In E. coli (PDB accessionnumber 3OAT),21 as well as in T. thermophilus (3OI3 not shown),20 the alkyl–aryl side chain of telithromycin (indicated by a blacktriangle) stacks upon the A752-U2609 base pair. (B) In D. radiodurans (1P9X)47 the side chain extends down the tunnel but doesnot come close to position 752. (C) In H. marismortui (1YIJ)39 the side chain folds over the macrolactone ring. The positions of thedesosamine sugar (indicated by open triangles) remain fairly invariant in different structures.

or for that matter in ribosomes of Gram-positivepathogens, remained a mystery until 2010 when theJ. Cate and T. Steitz laboratories reported the high-resolution structures of telithromycin complexed to70S ribosomes from E. coli and T. thermophilus.20,21

In these new structures, the aromatic head of thetelithromycin side chain closely approached the loopof helix 35 in domain II of 23S rRNA and establishedstacking interactions with the base pair formed be-tween A752 in this loop and U2609 in domain V(Fig. 4A). This interaction was in excellent agree-ment with the reported protection of A752 in the E.coli ribosome from DMS modification. Cate et al.noted that formation of the A752–U2609 base pairis impossible in H. marismortui where U2609 ofthe E. coli 23S rRNA is replaced with C, or in D.radiodurans, where C substitutes for A752.21 Im-portantly, the nucleotide sequences of 23S rRNAof many Gram-positive pathogens, including S. au-reus and S. pneumoniae are compatible with theformation of the A752–U2609 base pair, indicat-ing that the placement of the ketolide’s alkyl–arylside chain observed in the E70S and T70S struc-tures likely resembles its position in the ribosomesof clinical strains. Yet the exact position of the sidechain may slightly deviate in pathogens because theA752 protection observed in S. aureus was some-what less pronounced than that observed in E. coliribosomes.21

The structure and the site of attachment of thealkyl–aryl side chain to the macrolactone ring varyamong different ketolides. Nevertheless, in the E.

coli ribosomes, all the ketolides afford the samestrong protection of A752 from DMS modifica-tion, indicating that the placement of the side chainshould be fairly invariant. However, in D50S com-plexed with cethromycin, a ketolide structurallysimilar to telithromycin but carrying a distinctalkyl–aryl side chain at the C6 position rather thanat C11 as in telithromycin, the placement of theside chain notably deviates from that reported forthe telithromycin-D50S complex.47,48 In the D. ra-diodurans and H. marismortui ribosomes that lackthe A752–U2609 docking platform, the alkyl–arylchain apparently fails to lock in its proper place andadopts varying conformations that are likely irrele-vant to the placement of the drug in the ribosomesof Gram-positive pathogens.

The second azithromycin site inD. radiodurans

It is commonly assumed that most antibiotics havebeen evolutionarily selected to act upon one spe-cific target site. Surprisingly, crystallographic stud-ies have shown that some protein synthesis in-hibitors can bind to several sites in the ribosome.For example, aminoglycosides bind to helix 44 in16S rRNA as well as helix 69 in 23S rRNA of the E.coli ribosome,49 whereas up to five binding sites havebeen reported for tetracycline in the small subunit ofthe T. thermophilus ribosome.50,51 The existence ofsecondary antibiotic sites may simply stem from thelarge size of the ribosome, which offers many cavi-ties that provide a favorable geometry and chemical



Figure 5. Two azithromycin binding sites in the D. radioduransribosome.48 The first molecule of azithromycin (Azm I) bindsin the conventional macrolide binding site; the second molecule(Azm II) binds farther down the tunnel. The loops of proteinsL4 and L22 that form the tunnel constriction and contribute tothe Azm II binding are shown in blue. A hydrogen bond betweenAzm I and Azm II is shown by a dotted line.

environment for drug binding;52,53 binding of adrug in such sites is not expected to produce anyspecific effect on translation. On the other hand, itis conceivable that some antibiotics have been evo-lutionarily optimized for interacting with more thanone ribosomal site in order to achieve the full extentof their inhibitory action.

Biochemical and crystallographic analyses of in-teractions of macrolides with the E. coli ribosome,as well as with ribosomes of H. marismortui andT. thermophilus, consistently prove the existenceof a unique macrolide binding site located in theNPET near the PTC.19,39,54 Surprisingly, however,the binding of two azithromycin molecules was ob-served in the tunnel of the D. radiodurans large ribo-somal subunit48 (Fig. 5). In D50S, one azithromycinmolecule is found at the canonical macrolide sitewhere the central macrolactone ring of the drug,the desosamine and cladinose side chains assume aposition similar to that seen for macrolides in otherspecies. The second azithromycin molecule bindsin the D50S NPET next to the first one, but farther

away from the PTC, where it is inserted at the tunnelconstriction between the extensions of proteins L4and L22. The distinct amino acid sequences of theL4 and L22 proteins could potentially account forthe binding of the second azithromycin molecule inthe D. radiodurans ribosome. In the second site, thedrug interacts with the amino acid residues Tyr59,Gly60, Gly63, and Thr64 of L4 (D. radiodurans num-bering is used for the D50S ribosomal proteins). Ofthese, Gly60 is the least conserved and is frequentlyreplaced by Lys or Arg (or more rarely Pro or Ala).The presence of the bulkier amino acids at this L4position in the ribosomes of most bacteria could in-terfere with binding of azithromycin to the secondsite. Similarly, Arg111 of the L22 protein, with whichazithromycin interacts in its D50S second-bindingsite, is not well conserved and, thus, may accountfor the unusual drug binding properties of the D.radiodurans ribosome.

When bound in the neighboring sites in D50S,the two azithromycin molecules come close enoughto each other to establish a direct hydrogen bondinteraction between the N2 of the desosamine ofthe drug in the second site and O12 of the lactonering of azithromycin in the canonical binding site(Fig. 5). Hypothetically, such an interaction can leadto binding cooperativity, which could enhance theaffinity of the drug for the ribosome. A similar bind-ing of two drug molecules to D50S was also reportedfor bridged ketolides.55 While foot-printing studiesfailed to support the existence of a second-macrolidemolecule in the D50S,56 drug-binding experimentshave shown that the D. radiodurans ribosome canbind two macrolide molecules in solution.54 If, thebinding of two macrolides in the D50S NPET isconfirmed, it could open an interesting avenue foroptimizing macrolide drugs to stimulate their bind-ing to neighboring sites in the tunnel of ribosomesof bacterial pathogens.

Unusual binding of troleandomycin in theD. radiodurans large ribosomal subunit

Troleandomycin (triacetyloleandomycin) is a 14-member-ring macrolide derived by the acetylationof its natural precursor oleandomycin, at three sites:at the C12 position of the lactone ring, the 2′ positionof C3 oleandrose sugar, and the 4′ of the C5 aminosugar (Fig. 1A). In the crystallographic structureof troleandomycin complexed with D50S, the drugbinds in the NPET, however at a site significantly



Figure 6. Different binding modes of troleandomycin in thelarge ribosomal subunits of D. radiodurans and H. marismortui.In the H. marismortui ribosome (PDB accession number 3I56),58

the placement of troleandomycin (salmon) is similar to that ofother macrolides. In D. radiodurans (1OND),57 troleandomycinmolecule (green) is shifted down the tunnel and makes contactswith the amide nitrogen of Ala2 of ribosomal protein L32 (dottedline). The position of A2058 is shown for reference.

shifted “down” the tunnel relative to the conven-tional macrolide-binding site (Fig. 6).57 Binding oftroleandomycin to D50S was reported to promotethe rearrangement of �-hairpin of the L22 protein.57

In contrast to the D50S structure, in H50S, trolean-domycin binds at the conventional macrolide siteand does not reorient the L22 hairpin.58 In D50S,the binding of troleandomycin to an alternative sitecould be favored due to the formation of a hydro-gen bond between the Ala2 amide of the protein L32and the oxygen atoms of the C9 oxirane and theC10 keto-group of troleandomycin.57 Archeal ribo-somes do not contain an equivalent protein, whereasE. coli contains L32, but the protein lacks the firstfour amino acid residues that are present in the D.radiodurans L32 and thus may not support drugbinding in the alternative site.45

Species-specific effect of macrolideresistance mutations

Interspecies variations in the ribosome structure af-fect the binding of macrolides not only to wild-typeribosomes, but also to ribosomes that have acquiredresistance mutations. As a result, a mutation thatmakes one organism resistant to high concentra-tions of an antibiotic may only have a mild effect inanother species.

One of the most frequently found mutations thatrenders cells highly resistant to most 14- and 15-member ring macrolides, including ketolides, is anadenine to guanine transition at position 2058 in23S rRNA (see Vester et. al.59 for review). However,in S. pneumoniae, the A2058G mutation has little ef-fect on susceptibility to ketolides.46,60 The reason forthe unusual behavior of the S. pneumoniae A2058Gmutant was traced to the nature of the neighboring2057–2611 base pair that forms a part of the cradlehosting the macrolactone ring in the ribosome (seeFig. 2C).61 In many bacteria, including M. smeg-matis, the position 2057 is occupied by an adeninepaired to uridine at position 2611. In this back-ground, the A2058G mutation confers resistance toa high concentration of the ketolide, telithromycin.However, when the 2057A–2611U pair is replacedwith G–C, as in S. pneumoniae, the extent of ke-tolide resistance conferred by the A2058G mutationis significantly diminished. Presumably, when theantibiotic binds with high affinity to the wild-type(A2058) ribosome, the identity of the 2057–2611base pair does not play a major discriminatory role.However, when the drug binding is weakened bythe A2058G mutation, the nature of the base-pairedresidues 2057 and 2611 becomes important.

In addition to the direct effect of species-specificnucleotide differences upon antibiotic binding,variations in ribosome structure may affect the fit-ness cost of the resistance mutations and therefore,the prevalence of specific mutations in different or-ganisms. For example, in pneumococci, the A2059Gresistance mutation is observed far more frequentlythan the A2058G mutation, which is widespread inmajority of other bacterial species.59,62 The A2058Cmutation occurs frequently in Helicobacter pyloriand confers a high level of macrolide resistance;however, this mutation is deleterious in E. coli andthus usually does not show up in selection exper-iments.63,64 The A2058U mutation provides sig-nificant macrolide resistance in E. coli but onlymoderate resistance in H. pylori.63,65 Although noexhaustive analyses of protein mutations have beencarried out, different amino acid residues of pro-teins L22 and L4 are usually found to be mutatedin macrolide–resistant isolates of varying bacte-rial species (reviewed by Franceschi et al.62). Suchspecies-specific biases in occurrence of resistancemutations show that variations in the ribosomestructure may influence both, the fitness cost of



resistance as well as the differential binding of thedrug to the wild-type and mutant ribosomes in dif-ferent bacteria.

The role of the posttranscriptionalmodifications in species-specific mode ofmacrolide binding

A number of indigenous rRNA modifying enzymesmethylate or pseudouridylate-specific rRNA nu-cleotides at functionally important regions in theribosome. Although the mission of rRNA mod-ifications remains an enigma, it is generally be-lieved that they fine-tune the functions of the ri-bosome in translation. Approximately one-third ofmodified residues of the 23S rRNA are clusteredaround the NPET and some of them directly af-fect rRNA segments that constitute the macrolide-binding site. Posttranscriptional modifications thatalter the chemical makeup of the binding site mayhave an important influence upon macrolide bind-ing. One such heavily modified rRNA segment isin the loop of helix 35 in 23S rRNA, which inE. coli includes m1G745, �746, and m5U747 (seeRefs. 26,66–68)(Fig. 7). The modification patternvaries significantly between ribosomes of differ-ent bacterial species and influences antibiotic bind-ing.69 The enzyme RlmAI responsible for the N1methylation of G745 is found predominantly inGram-negative bacteria.26 In Gram-positive organ-isms, RlmAI is replaced with RlmAII, which addsa methyl group to N1 of the neighboring G748.70

When A2058 is monomethylated by the actionof an Erm enzyme, the methylation of G748 byRlmAII confers resistance to tylosin.26 Therefore,the A2058 monomethyltransferase would renderGram-positive bacteria that carry the rlmAII generesistant to tylosin, but would fail to do so withGram-negative organisms that lack rlmA.II

The loop of helix 35 in 23S rRNA is close to theNPET constriction formed by proteins L4 and L22.The macrolide resistance mutations in L22 alter theconformation of nucleotides 747 and 748.71 Thus,the natural diversity in the modification pattern ofthis rRNA segment may hypothetically modulatethe allosteric effects of the L22 and L4 mutations.

Species-specific difference in the post-transcriptional modification pattern in othersites of 23S rRNA may also affect the binding ofmacrolide antibiotics. For instance, the lack ofpseudouridylation of U2504 was shown to render

Figure 7. Posttranscriptional modifications in the loop of helix35 of 23S rRNA. Shown is the secondary structure of helix 35in the E. coli ribosomes where three positions, G745, U746, andU747 are modified to m1G, Ψ and m5U, respectively. Gram-positive bacteria usually carry m1G748 instead of m1G745.66,67

In some bacterial species, the loop of helix 35 is unmodified.69

Enzymes responsible for specific modifications are indicated.

E. coli hypersensitive to several PTC-targeting an-tibiotics.72 Therefore, it is expected that organismslacking the RluC enzyme responsible for U2504modification would exhibit hypersusceptibility to16-member-ring macrolides that reach into thePTC active site.

The variations in the NPET structure affectthe mode of action of macrolides

It has long been thought that macrolides in-hibit translation of all proteins because they“plug” the NPET. However, recent structural ev-idence shows that blocking of the tunnel by themacrolide molecule is incomplete.39 Some pro-teins can successfully thread through the tunnelnarrowed by the antibiotic and evade macrolideinhibition28,73 (Kannan et al., in preparation). Inthe conventional plug scenario, the structure of thetunnel would have little relevance to the mode ofmacrolide action. However, the ability of the nascentpolypeptide to slither through the narrowed crawlspace in the drug-obstructed NPET should be crit-ically influenced by the tunnel architecture, which



Figure 8. The variation in the ribosome tunnel structure between bacterial species. (A) The 23S rRNA residues located in theNPET and differing between Gram-negative E. coli and Gram-positive B. subtilis. Segments of 23S rRNA that form the walls of theexit tunnel are indicated by thick lines on the 23S rRNA secondary structure diagram and nucleotide variations are indicated byred 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-teria74 (Fig. 8).

We have recently discovered that bacterial cellstreated with very high (100-fold MIC) concentra-tions of erythromycin or telithromycin can still effi-ciently synthesize a subset of polypeptides (Kannanet al., in preparation). Importantly, the spectrum ofsuch “escape” proteins vary substantially betweenGram-negative E. coli and Gram-positive S. aureuslikely reflecting different architecture of the NPETsin the ribosomes of these organisms. The nature ofproteins that escape macrolide inhibition are likelyto influence cell growth and viability. Therefore, thevariation in the structure of the ribosomal tunnelin the vicinity of the macrolide binding site couldbe a major contributing factor to the differential ef-fect of specific macrolide antibiotics upon differentbacterial species.

Concluding remarks

Here, we presented several examples illustratinghow macrolide binding and action, as well as bac-terial resistance to these antibiotics, can be affectedby the species-specific traits of ribosome structure.There is no doubt that the use of model organ-isms was incredibly important for understandingthe basic principles of binding and action of thesedrugs. However, structure-guided efforts for the de-velopment of novel antibiotics need to take into ac-count the interspecies variations in ribosome struc-ture and possible differences in drug binding sites in

ribosomes of pathogenic strains. The first efforts inthis direction illustrate the promise of structure-based drug design.75 Expanding the number ofspecies from which crystallizable ribosomes can beobtained, while ensuring that new structures remainin the public domain, could significantly acceleratedevelopment of new, useful antibiotics and/or stim-ulate novel applications of known drugs.

Acknowledgments

We thank Lisa K. Smith and Jacqueline M. LaMarrefor proofreading the manuscript and Artem Mel-man for verifying the antibiotic structures. The workrelated to the sites of antibiotic action in this lab-oratory is supported by a grant from the UnitedStates-Israel Binational Science Foundation (No.2007453).

Conflicts of interest

The authors declare no conflicts of interest.

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