Download On the specificity of antibiotics targeting the large ribosomal subunit

Ann. N.Y. Acad. Sci. ISSN 0077-8923
A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S
Issue: Antimicrobial Therapeutics Reviews
On the specificity of antibiotics targeting the large
ribosomal subunit
Daniel N. Wilson1,2
1
Center for integrated Protein Science Munich (CiPSM), Germany. 2 Gene Center and Department of Biochemistry,
Ludwig-Maximilians-Universität München, Feodor-Lynenstr 25. München, Germany
Address for correspondence: Daniel N. Wilson, Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität
München, Feodor-Lynenstr. 25, 81377 München, Germany. [email protected]
The peptidyltransferase center of the large ribosomal subunit is responsible for catalyzing peptide bonds. This active
site is the target of a variety of diverse antibiotics, many of which are used clinically. The past decade has seen a
plethora of structures of antibiotics in complex with the large ribosomal subunit, providing unprecedented insight
into the mechanism of action of these inhibitors. Ten distinct antibiotics (chloramphenicol, clindamycin, linezolid,
tiamulin, sparsomycin, and five macrolides) have been crystallized in complex with four distinct ribosomal species,
three bacterial, and one archaeal. This review aims to compare these structures in order to provide insight into the
conserved and species-specific modes of interaction for particular members of each class of antibiotics. Coupled
with the wealth of biochemical data, a picture is emerging defining the specific functional states of the ribosome
that antibiotics preferentially target. Such mechanistic insight into antibiotic inhibition will be important for the
development of the next generation of antimicrobial agents.
Keywords: antibiotics; protein synthesis; ribosome; RNA; species specificity; translation; X-ray crystallography
Introduction
The synthesis of proteins in the cell occurs on
large macromolecular complexes called ribosomes
(reviewed by Schmeing and Ramakrishan1 ). Ribosomes provide the platform upon which the codons
of the mRNA are decoded by the anticodons of the
tRNAs. In this way, tRNAs can deliver the appropriate amino acid to the ribosome so that it can
be incorporated into the growing nascent polypeptide chain. The ribosome has three tRNA binding
sites, the A, P, and E sites. The A site is where incoming aminoacyl-tRNA (aa-tRNA) enters the ribosome. Prior to peptide bond formation, the P site
contains the peptidyl-tRNA, that is, the tRNA bearing the polypeptide chain. The E site binds exclusively deacylated or uncharged tRNAs, that is, those
tRNAs that have incorporated their amino acid into
the polypeptide chain and are ready to exit from
the ribosome. Thus, during translation the tRNAs
move from A→P→E. Peptide-bond formation occurs at the peptidyltransferase center (PTC) of the
large ribosomal subunit (Fig. 1A) and requires the
accurate positioning of the 3 terminal CCA-ends of
the aa-tRNA at the A site and peptidyl-tRNA in the
P site. The ribosome represents a major target in the
cell for antibiotics, with many clinically used antibiotics that interfere with the process of peptide-bond
formation.2
The wealth of decades of biochemical studies on
antibiotic action on ribosomes can now be interpreted in the light of the crystal structures of the ribosome in different functional states and in complex
with many different classes of antibiotics (reviewed
by Wilson3 ). Indeed, multiple crystal structures of
the same antibiotic in complex with ribosomal particles from a variety of different species have been determined (Table 1). Comparisons of such structures
have already provided some surprising differences in
the manner by which the same member of an antibiotic class interacts with distinct ribosomal particles.4
Here the aim is to provide an update on the diverse
modes of antibiotic interaction with the large subunit, by comparing the available X-ray structures
doi: 10.1111/j.1749-6632.2011.06192.x
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 1
Antibiotics targeting the large ribosomal subunit
Wilson
Figure 1. Chloramphenicol (CAM) binds at the A site of the PTC. (A) Overview of the large subunit, showing relative positions of
P-tRNA (green), A site (pink), polypeptide chain (tan), and ribosomal proteins L1 (blue) and L11 (purple). (B) Chemical structure
of CAM. (C) Binding position of CAM-16 (gold) and CAM-29 (cyan) relative to A- (blue) and P-tRNA (green).74 (D) Comparison
of CAM1 from D50S,8 E70S,6 and T70S5 structures. (E) Binding position of CAM1-E70S6 (gold), CAM1-T70S5 (orange), CAM-29
(cyan), and ERY-E70S6 (tan) relative to A-tRNA74 (blue) and TnaC peptidyl-tRNA27 (green).
of antibiotics that have been crystallized in complex with the large ribosomal subunit from different
species, namely the chloramphenicols, macrolides,
oxazolidinones, pleuromutilins, and sparsomycins
bound to the large 50S subunit of Deinococcus radiodurans (D50S) and Haloarcula marismortui (H50S)
as well as the 70S ribosome of Escherichia coli (E70S)
or Thermus thermophilus (T70S) (Table 1). Understanding how variations in the drug composition,
as well as the drug target, can influence the binding
and inhibitory activity of antibiotics will not only
provide insight into the mechanism of drug action,
but will also be important for future development
of new improved antimicrobial agents.
The orientation of chloramphenicol at the
A site
Chloramphenicol (CAM), originally isolated from
Streptomyces venezuelae, consists of a para-
2
nitrophenyl ring attached to a dichloroacetamido
tail (Fig. 1B). CAM displays broad-spectrum activity, inhibiting a wide range of Gram-positive and
-negative bacteria, but not translation on eukaryotic
cytosolic ribosomes (reviewed by Wilson3 ). CAM
has been crystallized in complex with three bacterial
ribosomal particles (Table 1), revealing a primary
CAM-1 binding site at the PTC (Fig. 1C). In the
T70S5 and E70S structures,6 the phenyl ring binds
analogously and planar to a phenyl-moiety of an
A-tRNA (Fig. 1C), consistent with the observation
that CAM interferes with the puromycin reaction as
well as the binding of small tRNA fragments to the
A site of the PTC.7 An earlier CAM-D50S structure
at lower resolution8 reported CAM-1 with a different orientation, rotated by ∼180◦ , with respect to
the CAM-T70S/E70S structures (Fig. 1D). Furthermore, the phenyl ring was orthogonal to the plane
of the phenyl-moiety of the A-tRNA. In contrast, no
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 Wilson
Antibiotics targeting the large ribosomal subunit
Table 1. Structures of similar antibiotic–ribosome complexes available from different species
Antibiotic
Chloramphenicols
Chloramphenicol
Chloramphenicol
Chloramphenicol
Chloramphenicol
Lincosamides
Clindamycin
Clindamycin
Clindamycin
Macrolide/ketolides
Azithromycin
Azithromycin
Azithromycin
Azithromycin
Carbomycin A
Carbomycin (Josamycin)
Erythromycin
Erythromycin
Erythromycin
Erythromycin
Erythromycylamine
Lankamycin
Methymycin
Rapamycin
RU-69874
Solithromycin (CEM-101)
Telithromycin
Telithromycin
Telithromycin
Telithromycin
Triacyloleandomycin
Troleandromycin
Tylosin
Oxazolidinones
Linezolid
Linezolid
Pleuromutilins
Tiamulin
Tiamulin
Sparsomycin
Sparsomycin
Sparsomycin
Complex
Species
Res (Å)
PDB ID
Reference
50S subunit
50S subunit
70S ribosome
70S ribosome
D.r
H.m
E.c
T.t
3.5
3.0
3.2
3.0
1K01
1NJI
3OFA-D
3OGE/Y, 3OH5/7
1
2
3
4
50S subunit
70S ribosome
50S subunit–G2099A
D.r
E.c
H.m
3.1
3.2
3.0
1JZX
3OFX-Z, 3OG0
1YJN
1
3
5
50S subunit
50S subunit
50S subunit–G2099A
70S ribosome
50S subunit
50S subunit
50S subunit
50S subunit–G2099A
70S ribosome
70S ribosome
50S subunit
50S subunit
50S subunit
50S subunit
50S subunit
70S ribosome
50S subunit
50S subunit–G2099A
70S ribosome
70S ribosome
50S subunit
50S subunit
50S subunit
D.r
H.m
H.m
T.t
H.m
D.r
D.r
H.m
E.c
T.t
D.r
D.r
D.r
D.r
D.r
E.c
D.r
H.m
E.c
T.t
H.m
D.r
H.m
3.2
3.2
2.4
3.0
3.0
3.3
3.5
2.7
3.1
3.0
3.6
3.3
3.7
3.8
3.6
3.3
3.4
2.6
3.3
3.1
2.9
3.4
3.0
1NWY
1M1K
1YHQ
3OHY/Z/0/1
1K8A
2O44
1JZY
1YI2
3OFO-R
3OHC/D/J/K
2O43
3PIO
3FWO
1Z58
2O45
1VT2, 3OR9/A/B
1P9X
1YIJ
3OAQ-T
3OI2-5
3I56
1OND
1K9M
6
7
5
4
7
8
1
5
3
4
8
9
10
11
8
12
13
5
3
4
14
15
7
50S subunit
50S subunit
D.r
H.m
3.5
2.7
3DLL
3CPW
16
17
50S subunit
50S subunit
D.r
H.m
3.5
3.2
1XBP
3G4S
6
18
50S subunit
50S subunit
D.r
H.m
3.5
3.2
1NJM/N
1M90, 1VQ8/9
19
2, 20
T.t, Thermus thermophilus; D.r, Deinococcus radiodurans; E.c, Escherichia coli ; H.m, Haloarcula marismortui.
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 3
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Wilson
CAM-1 binding site was observed in H50S structures solved with 20 mM CAM; however, a second
binding site (CAM-2) was detected deeper within
the tunnel, overlapping the binding position of the
macrolide erythromycin (ERY) (Fig. 1E).9 Equilibrium dialysis studies reported two binding sites for
CAM on bacterial ribosomes, one with high affinity
(K d 2 ␮M) and the other of low affinity (K d 200
␮M),10 which could reflect the CAM-1 and CAM-2
sites, respectively. Although no evidence for CAM-2
was seen in the E70S structure determined using 0.5
mM CAM,6 cross-linking of CAM to E. coli and archaeal H. halobium ribosomes identified modifications within the ERY binding site consistent with the
CAM-2 position.11 Nevertheless, it seems unlikely
that the CAM-2 site is critical for the inhibitory
mechanism of the drug because of its low affinity and that most of the CAM resistance mutations
and modifications cluster around CAM-1 within the
A site of the PTC.3,12
CAM is well known as an elongation inhibitor
since addition of the drug to growing bacterial cells
stabilizes polysomes. The inhibitory action of CAM
is dependent on the nature of the substrates: translation of synthetic mRNAs encoding bulky aromatic
side chains, such as poly(U) for Phe, or poly(UA)
for Tyr/Ile, is less effectively inhibited by CAM than
translation of mRNAs encoding smaller or charged
amino acids, such as poly(A) for Lys, poly(C) for
Pro, or poly(UC) for Ser/Leu (reviewed by Pestka13 ).
Similarly, higher concentrations of CAM are required to inhibit the ribosome binding of the tRNA
fragments, such as CCA-Phe, compared to CCALys, CCA-Leu, or CCA-Ser.10 Collectively, these data
suggest that some aa-tRNAs compete better with
CAM due to their higher affinity for the A site of
the ribosome. The nature of the P site substrate
also influences CAM activity: CAM inhibits the
puromycin reaction with AcPhe-tRNA in the P site,
but not with Ac(Phe)2 -tRNA and CAM can even
induce dissociation of Ac(Phe)2–4 -tRNAs from the
ribosome,14 suggesting that the polypeptide chain
attached to the P site can antagonistically influence
activity of CAM bound in the A site. In contrast,
CAM can also act synergistically with the P site substrate and has been shown to stimulate the binding of CACCA-NAcLeu fragments to the P site of
the ribosome.15 Thiamphenicol (an analog of CAM
where the para-nitro moiety is replaced by a methylsulfonyl moiety) is also reported to stabilize the
4
binding of fMet- and AcPhe-tRNAs to the P site
of the ribosome.16 These data are consistent with
the CAM-1 position from the E70S (or T70S) structures5,6 where the para-nitro/methyl-sulfonyl moiety of CAM/thiamphenicol is in close proximity to
the aminoacyl moiety of the P-tRNA.
Linezolid and interaction with the P-tRNA
Linezolid (LIN), a synthetic compound belonging
to the oxazolidinone class of antibiotics, is used
clinically to treat a variety of Gram-positive infections (reviewed by Leach17 ). LIN comprises three
aromatic rings with an acetamidomethyl tail attached to the pharmacokinetic oxazolidinone ring A
(Fig. 2A). A wealth of biochemical and structural
evidence (reviewed by Wilson3 ) indicates that LIN
binds at the PTC of the large subunit, in a position
overlapping with the aminoacyl-moiety of an A site
bound tRNA (Fig. 2B). There is an excellent overall agreement in the LIN binding position derived
from cross-linking data18 and subsequently visualized using X-ray crystallography (Fig. 2C).19,20 In
each case, the acetamidomethyl tail of LIN extends
down the tunnel toward A2503 (E. coli numbering
is used throughout) of the 23S rRNA, whereas the
morpholino ring C approaches U2585 (Fig. 2D).
Differences are nevertheless observed when comparing the structures of the bacterial LIN-D50S20
and archaeal LIN-H50S19 structures. First, the fluorophenol ring B of LIN is rotated by ∼70◦ relative to
the oxazolidinone ring A between the two structures
(Fig. 2C). The outcome being that in the LIN-D50S
structure the fluorophenyl ring is sandwiched between A2451 and U2506, whereas in the LIN-H50S
the same ring stacks on C2452. This may result partially from the shifted position of U2506 in D50S,
which would clash with LIN-H50S binding position (Fig. 2D). But the difference could also be due
to the presence of the CCA-Phe bound in the P site
of the H50S-LIN structure since it would encroach
on the fluorine atom of LIN in the D50S structure
(Fig. 2D). Second, differences are observed in the location and flexibility of U2504 between bacterial and
archaeal ribosome structures: in the former, U2504
is observed stacking upon the oxazolidinone ring
A of LIN. In contrast, an alternate conformation is
observed for U2504 in archaeal (H50S) structures,
except when LIN is bound. This indicates that binding of LIN to H50S reorients U2504 to allow stacking with the oxazolidinone ring of LIN as seen in
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 Wilson
Antibiotics targeting the large ribosomal subunit
Figure 2. Linezolid (LIN) binds at the A site of the PTC. (A) Chemical structure of LIN. (B) Binding position of LIN20 (gold),
relative to A- (blue) and P-tRNA (green).74 (C) Comparison of LIN from D50S,20 H50S,19 and E50S18 structures. (D) Binding
position of LIN at the PTC of D50S,20 (gold) and H50S (blue) with P-tRNA74 (green). (E) Comparison of 23S rRNA nucleotides
neighboring U2504: bacteria have A2572 (yellow-orange), whereas archaea have U2572 (blue). Arrows indicates different position
of U2504 in native H50S75 (light blue) versus LIN-H50S19 (dark blue).
the LIN-D50S (arrowed in Fig. 2E). In bacteria, the
presence of a bulkier adenine at position 2572 (instead of uridine, as in H50S) would prevent U2504
from rotating back (Fig. 2E).19 High concentrations
(5 mM) of LIN were used in the LIN-H50S structure, which may have been necessary to mobilize
U2504,19 whereas 1,000-fold lower concentrations
(5 ␮M) were used for the bacterial structure.20 Surprisingly, however, the archaea Halobacterium halobium is reported as being sensitive to LIN, with a
minimal inhibitory concentration of 3 ␮M,21 making it unclear why such high LIN concentrations
were used for H50S.
Despite the detailed structural characterization of
LIN on the ribosome, the exact mechanism of action
of oxazolidinones still remains unclear. Based on the
binding position, LIN should prevent correct placement of the aminoacyl-moiety of the A-tRNA and in
doing so inhibit peptide-bond formation. Surprisingly, there are conflicting reports as to the ability
of different oxazolidinone members to inhibit the
puromycin reaction.3 Moreover, when inhibition
is reported, nonphysiologically high concentrations
of a drug (∼1 mM) are required. Similarly, while
oxazolidinones are observed to compete effectively
for ribosome binding with CAM, lincosamides, and
puromycins, the IC50 s are in the ∼1 mM range.22,23
In contrast, the IC50 for LIN determined using in
vitro translation systems is significantly lower (∼1–
10 ␮M),23–25 suggesting that LIN targets a particular
functional state of the ribosome. In this respect it
is interesting that Ippolito et al.19 report that the
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 5
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Wilson
quality of the electron density for LIN was superior in the presence of an aminoacyl-tRNA mimic
CCA-Phe, suggesting that the interaction with the
P-tRNA increases the affinity of LIN for the ribosome. Indeed, oxazolidinones have been reported
to crosslink to small tRNA-sized molecules on
the ribosome.26 Superimposition of the recently
determined structure of ribosome stalled with a
nascent polypeptide chain attached to the P-tRNA,27
predicts that LIN would also interact with the
C-terminal 2–4 amino acids of the nascent chain
(Fig. 2B). It will be interesting to examine the influence of the nature of the aminoacyl or peptidy
moiety of the P-tRNA on the binding and inhibition
of oxazolidinones in order to define exactly which
functional state is targeted by this class of antibiotics.
The orientation of the clindamycin tail
and A site inhibition
Clindamycin (CLN) is a semisynthetic lincosamide
that contains a galactose sugar linked to a propylpyrrolidinyl moiety (Fig. 3A). CLN is active against
most Gram-positive bacteria as well as some protozoa, such as Plasmodium falciparum (reviewed by
Spizek28 ). Crystal structures of CLN bound to bacterial (D50S and E70S)6,8 and archaeal (H50S)29
ribosomes (Table 1) locate the drug to the A site of
the PTC (Fig. 3B). The galactose sugar of CLN is
Figure 3. Clindamycin (CLN) binds at the A site of the PTC. (A) Chemical structure of CLN. (B) CLN bound to D50S8 (pink),
H50S29 (orange), and E70S6 (gold), relative to A- (blue) and P-tRNA (green).74 (C) Binding position of CLN-E70S6 (gold), and
ERY-E70S6 (tan) relative to A-tRNA74 (blue) and TnaC peptidyl-tRNA27 (green). (D) Conformation of 23S rRNA nucleotides
comprising the CLN binding pocket of D50S8 (pink), H50S29 (orange), and E70S6 (gold). The arrow indicates the different position
of the propyl-tail of CLN in D50S versus E70S and H50S.
6
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 Wilson
Antibiotics targeting the large ribosomal subunit
Figure 4. Sparsomycin (SPAR) stabilizes the P-tRNA at the PTC. (A) Chemical structure of SPAR. (B) SPAR (orange) bound to
H50S9 (yellow), relative to A- (blue) and P-tRNA (green).74 Difference of A2602 conformation in SPAR-H50S (yellow) and PRE-state
H50S (cyan) is arrowed. (C) as in (B), but including SPAR (blue) and A2602 (light blue) from the SPAR-D50S structure.36
located in a position approaching the binding position of the desosamine sugar of the macrolide ERY,
whereas the pyrrolidinyl moiety overlaps the binding site of CAM as well as the aminoacyl moiety
of an A-tRNA (Fig. 3C). Consistently, lincosamides
have been shown to directly inhibit the transfer of
fMet or AcPhe to puromycin30,31 as well as compete
with both CAM and ERY for ribosome binding.32
The galactose sugar of CLN is located similarly in all
three structures,6,8,29 where it interacts with A2503,
A2058, and A2059 (Fig. 3D), providing an explanation as to how modification or mutation of these nucleotides can confer resistance to lincosamides.12,33
In contrast, the propyl-pyrrolidinyl moiety of CLN
in the D50S structure8 is rotated by ∼90◦ when compared with the H50S29 and E70S6 structures (Fig. 3B
and D). As stated previously,4 the lack of density for
the propyl extension of CLN in the D50S structure led to a placement based on the available small
molecule structure, and it was also noted that the
conformation of pyrrolidinyl moiety of CLN as reported for the H50S structure would fit the density
equally well. Support for the H50S/E70S conformation comes, first, from the orientation of U2504
that is invariant in the available bacterial structures
and, second, from observation that the CLN-D50S
position8 requires some adjustment to U2504 and
C2452 to accommodate the propyl tail (Fig. 3D).
Nevertheless, the poor density for the propyl tail in
the D50S structure, suggests that it is highly flexible
and therefore may contribute less to the binding of
CLN to the ribosome. From a drug–design perspective, this region of the molecule may provide avenues
for modification to develop successive generations
of lincosamide antibiotics with improved ribosome
interactions and/or pharmacological properties.
Sparsomycin interaction with A2602
promotes translocation
Sparsomycin (SPAR), a nucleoside analog of uracil
(Fig. 4A) produced by Streptomyces sparsogenes,
is a potent inhibitor of peptidyltransferase activity in bacteria, archaea, and eukaryotes (reviewed
by Lazaro et al.34 ). SPAR has been crystallized together with tRNA mimics in complex with both archaeal (H50S)9,35 and bacterial (D50S)36 ribosomes
(Table 1). In both cases, although the uracil moiety
of SPAR was seen to stack upon A2602, consistent
with previous crosslinking data,37 dramatic differences in the overall SPAR binding positions were
observed (Fig. 4B and C). Bound to H50S, SPAR
stacks between A2602 and the P site CCA-Phe analog (Fig. 4B), consistent with the observation that
SPAR requires a P site substrate for ribosome binding.38 Binding of the CCA-Phe analogs normally
distributes evenly between the A and P sites in the
H50S crystals, but in the presence of SPAR, CCAPhe occupies only the P site.9,35 This is understandable since the conjugated tail of SPAR overlaps with
the aminoacyl-moiety of an A-tRNA (Fig. 4B) and
thus would prevent binding not only of A-tRNA,
but also CAM and lincomycin in agreement with
previous reports.34,39 The binding site of SPAR in
the D50S structures in contrast spans across the P
site and does not encroach on the A site (Fig. 4C).36
Although SPAR was also crystallized together with
a tRNA substrate in the D50S structure, no interaction was observed between SPAR and the tRNA
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 7
Antibiotics targeting the large ribosomal subunit
Wilson
substrate. In fact, an identical SPAR binding position was observed in the absence of the tRNA substrate.36 The SPAR-D50S binding position is hard
to reconcile with the ability of SPAR to stabilize the
P site substrate, since the reported binding position
would clash with P-tRNA (Fig. 4C). Unlike the H50S
crystals, binding of the tRNA substrates in the D50S
crystals appears to favor the A site,36 which may
have precluded formation of the stable functional
state observed in the H50S structure. Pretranslocation (PRE) state ribosomes with tRNAs in both
A and P sites are not protected from the action of
SPAR, since SPAR can induce translocation of the
tRNAs with the outcome that the peptidyl-tRNA is
stabilized at the P site and the A site is blocked by
the drug.40 A2602 is at the center of the rotational
symmetry of the PTC where it has been proposed to
play a role in guiding the CCA-end of the A-tRNA
to P site during translocation.41 SPAR appears to
induce a rotation of A2602 compared to the PRE
state conformation (Fig. 4B), which may reflect the
two-step reaction mechanism of SPAR—a slow ini-
tial step that isomerizes slowly to adopt a more stable conformation.42 In this regard, the position of
SPAR observed in the D50S structure (Fig. 3C) may
reflect an initial binding event, postpeptide bond
formation, where the deacylated tRNA is driven by
SPAR from the P to the E site. Subsequently, the
peptidyl-tRNA can then move into the P site where
it is stabilized by SPAR, as observed in the H50S
structures9,35 (Fig. 2B).
Pleuromutilins overlap both the A and P
sites at the PTC
Pleuromutilin was discovered in the 1950s as a natural product of two basidiomycete species, Pleurotus mutilis and Pleurotus passeckerianus, and displays activity against Staphlococcus aureus strains
(reviewed by Novak43 ). Modification of the pleuromutilin C14 tail led to the development of
semi-synthetic derivatives, such as tiamulin (TIA,
Fig. 5A),44 which was approved for veterinary usage
in the late 1970s. TIA has been crystallized in complex with bacterial (D50S)45 and archaeal (H50S)46
Figure 5. Tiamulin (TIA) binds across the A and P sites at the PTC. (A) Chemical structure of TIA. (B) TIA bound to D50S45 (gold)
and H50S46 (blue), relative to A- (blue) and P-tRNA (green).74 (C) TIA bound to D50S45 (gold), relative to A-tRNA74 (blue) and
TnaC peptidyl-tRNA27 (green). (D) Conformation of 23S rRNA nucleotides comprising the TIA binding pocket of D50S45 (gold)
and H50S46 (blue). The arrow indicates the different position of U2504 in D50S versus H50S. (E) as (D), but including additional
pleuromutilin structures.49
8
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Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 Wilson
large subunits (Table 1). These structures reveal that
the tricyclic mutilin core of TIA (Fig. 5A) overlaps
the binding site of aminoacyl-moiety of an A-tRNA
(Fig. 5B and C), consistent with the finding that TIA
competes with CAM and puromycin for ribosome
binding and prevents binding of aa-tRNAs to the
A site.47,48 In both structures, the C14-tail of TIA
extends toward the P site, although there is some
difference in the extent of overlap with the P-tRNA
(Fig. 5B and C). This distinction results partially
from the shifted position of the mutilin core, which
is located deeper in the A site of the H50S, but
mainly from the more extended conformation of
the C14-tail of TIA in the D50S structure (Fig. 5C).
In TIA-D50S, U2504 make an important contribution to the binding pocket of the mutilin core of
TIA, whereas in the H50S structure the equivalent
base is shifted away. This probably explains why 1
mM TIA was used for TIA-H50S,46 compared to 10
␮M (100× less) in TIA-D50S.45 Nevertheless, the
shifted position of U2504 is unlikely to account for
the shift in location of the mutilin core between
TIA-H50S and TIA-D50S, since subsequent structures of other pleuromutilin derivatives bound to
D50S49 reveal identical mutilin core placements as
in the TIA-H50S, yet the U2504 maintains a similar
confirmation to that in TIA-D50S (Fig. 5D).
Biochemical analyses indicate that TIA can destabilize binding of fMet-tRNA to the ribosome.47,50
The structures indicate that the C14-tail could perturb the placement of the aminoacyl-moiety of the
P-tRNA, accounting for tRNA drop-off. However,
the destabilizing effect is modest, indicating that simultaneous cohabitation of the initiator tRNA and
TIA may occur. Nevertheless, any initiation complexes that do form are nonproductive, since TIA
is a potent peptidyltransferase inhibitor by preventing A-tRNA binding.47 Pleuromutilins cannot,
however, inhibit translating ribosomes,50 suggesting that binding of TIA to ribosomes containing
peptidyl-tRNAs is prevented. This is easy to envisage
since (i) TIA binding, in particular the accommodation of the C14-tail, would be sterically blocked
by the presence of a polypeptide chain (Fig. 5C),
and furthermore (ii) the polypeptide chain in the
tunnel stabilizes the P-tRNA, preventing peptidyltRNA drop-off. Thus, the mechanism of action of
pleuromutilins is likely to be, on one hand, to prevent initiation complex formation by perturbing the
stable binding of the fMet-tRNA to the P site—an
Antibiotics targeting the large ribosomal subunit
ability that is likely to be influenced by the length
and nature of the C14-tail of the pleuromutilin, and
on the other hand, if initiation complex formation
does occur, then the A site location of the mutilin
core would prevent delivery of the aa-tRNA to the
A site and thus formation of the first peptide bond.
Such a model is in agreement with the observation
that addition of pleuromutilins to intact cells causes
a loss of polysomes and a concomitant stabilization
of 70S monosomes.50
The influence of species specificity on
macrolide-ribosome interaction
Macrolides represent a large class of polyketide compounds synthesized by actinomycetes, which inhibit
protein synthesis on eubacterial, but not archaeal
or eukaryotic ribosomes.51,52 Macrolide antibiotics
bind adjacent to PTC, within the tunnel through
which the polypeptide chain traverses during translation (Fig. 6A). The binding site of macrolides is vacant on free or initiating ribosomes, but unavailable
in elongating ribosomes.53,54 Generally, the presence of macrolide antibiotics within the ribosomal
tunnel restricts protein synthesis to short oligopeptides, which eventually dissociate from the ribosome
in the form of oligopeptidyl-tRNAs.55–57 However,
recent evidence has indicated that the inhibitory effect of macrolides appears to be also dependent on
the sequence of the nascent polypeptide chain, such
that some sequences can even escape the inhibitory
effect of the drug.58
Clinically used macrolides have 14-, 15-, or
16-membered lactone rings to which amino sugars
are attached at varying positions. For example,
the macrolide ERY has a 14-membered ring with
cladinose and desosamine sugars attached at the C3
and C5 positions, respectively (Fig. 6B). To date,
there are X-ray structures of ERY bound to four
different ribosomal particles; three bacterial (E70S,
T70S, and D50S) and one archaeal (H50S) (Table 1).
Comparison of the structures reveals that the binding position of ERY is identical in the T70S,5 E70S6
and H50S29 complexes (Fig. 6C). Within the limits
of the resolution, the conformation and placement
of the lactone ring and amino sugars as well as the
interactions established with the ribosome appear
to be conserved. This contrasts with the ERY-D50S8
and chemical similar erythromycylamine-D50S
(ERC-D50S)59 structures, where the lactone ring
conformations are markedly divergent (Fig. 6C).
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 9
Antibiotics targeting the large ribosomal subunit
Wilson
Figure 6. The macrolides ERY and TAO bind in the ribosomal tunnel. (A) Transverse section of the large subunit showing the
ribosomal tunnel and the relative positions of P-tRNA (green), macrolide (red), and path of the polypeptide chain (tan). (B–C)
Chemical structures of ERY and TAO. (C–D) Comparison of the binding positions of ERY bound to D50S (salmon), E70S6 (gold),
T70S5 (tan), and H50S29 (cyan) with erythromycylamine59 (ERC, pink), lankamycin60 (LNK, green), and RU6987459 (RUM, blue)
bound to the D50S. (E) Comparison of the binding positions of ERY bound to E70S6 (gold) with TAO bound to D50S62 (pink) and
H50S61 (cyan). The arrow indicates the difference between L22 in the native D50S (ribbon)76 and TAO-D50S62 (ball-trace).
This is particularly surprising since subsequent
D50S structures of the 14-membered macrolides
RU69874 (RUM)59 and lankamycin (LNK)60
are nearly identical to the ERY-E70S structure
(Fig. 6D). This observation is consistent with
the re-examination of the ERY-D50S at higher
resolution, which indicated that the conformation
of lactone ring of ERY-D50S is in fact similar to that
reported in the ERY-H50S structure.4 What was
not addressed is the conformation of 14-membered
macrolide troleandomycin (TAO). TAO is chemically similar to ERY (Fig. 6B) and, as expected,
binds to H50S61 in a very similar fashion to all other
14-membered macrolides, e.g., ERY (Fig. 6E). In
contrast, it is hard to rationalize why a completely
different TAO binding site is reported in D50S
structure.62 In D50S, TAO is reported to be located
10
deeper in the tunnel, with the C11-acetylation
overlapping the Arg111 sidechain of ribosomal
protein L22.62 This steric clash is suggested to be
sufficient to completely destabilize the tip of the
␤-hairpin of L22, causing it to flip into the lumen of
the tunnel (Fig. 6E).62 The flipped L22 position was
proposed to play a role in translational stalling;62
however, no such conformational change in L22 is
evident in the recent structures of translationally
stalled ribosomes.27,63,64 Nevertheless, detachment
of the tip of L22 has been observed when three amino
acids are deleted in L17 (L22 homolog) of H50S.29
The emergence of bacterial resistance to
macrolide antibiotics has led to the development of ketolides, such as telithromycin (TEL),
which are semisynthetic derivatives of macrolides
where the C3-sugar is replaced with a keto group
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 Wilson
Antibiotics targeting the large ribosomal subunit
Figure 7. Binding of TEL and AZI within the ribosomal tunnel. (A) Chemical structure of TEL. (B–C) Comparison of the binding
positions of ERY-E70S6 (gold) with (B) TEL bound to E70S6 (blue) and H50S29 (cyan), and (C) TEL bound to T70S5 (green) and
D50S65 (salmon). (D) Chemical structure of AZI. (E–F) Comparison of the binding positions of AZI-T70S5 (orange) with (E)
ERY-E70S6 (gold) and AZI-H50S29 (cyan) and (F) AZI-D50S69 (salmon). (G) Binding positions of AZI-1 and AZI-2 in D50S69 (red),
relative to L22 (orange).
(Fig. 7A). The broader spectrum of activity of
the ketolides is also related to the presence of
additional modifications and side chains, such
as the alkyl–aryl sidechain of TEL (Fig. 7A).
TEL has been crystallized in complex with the
D50S,65 H50S,29 T70S,5 and E70S6 (Table 1).
Comparison of TEL in the H50S, E70S, and T70S
structures indicates that the lactone ring and desosamine sugar are positioned in an identical fashion
as ERY bound to E70S (Fig. 7B and C). Although
the lactone ring of the TEL in the D50S structure
appears to deviate significantly,65 reexamination of
the TEL-D50S structure at higher resolution indicated that the conformation of lactone ring is in
fact similar to that reported in the TEL-H50S structure.4 In contrast to the lactone ring, the placement
of the alkyl–aryl side chain of TEL differs dramatically between different species: bound to the archaeal H50S,4 the TEL-side chain folds back across
the lactone ring and stacks upon C2609 whereas in
the bacterial E70S6 the side chain of TEL is rotated
by 120◦ and stacks upon the A752-U2609 base-pair.
This Watson–Crick base-pair cannot form in H50S
because the equivalent bases to A752 and U2609
are both pyrimidines, providing an explanation for
the alternate position in H50S. A similar stacking arrangement of the alkyl–aryl side chain is also seen for
the fluoroketolide solithromycin (CEM-101) bound
to the E70S66 as well as for TEL bound to T70S
(Fig. 7C).5 Like H50S, D50S also has two pyrimidines at the equivalent positions to 752 and 2609;
however, unlike H50S, the side chain of TEL does
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 11
Antibiotics targeting the large ribosomal subunit
Wilson
not stack upon U2609, but rather adopts a unique
conformation contacting C790 (Fig. 7C).65 In H50S,
the equivalent base to C790 is rotated away and unavailable for interaction (arrowed in Fig. 7C). While
C790 is available for stacking in E70S and T70S, the
interaction with A752-U2609 appears to be energetically more favorable. Similar interactions with the
A752-U2609 are predicted for the Kosan ketolide K1325, where the alkyl–aryl side chain is attached to
the lactone ring at the C13 position.67 Thus, in contrast to the lactone ring, the attached heterocyclic
side chain of TEL interacts with fewer conserved regions of the ribosome, which allows distinct conformations to be adopted across different organisms,
and even between distinct bacterial species.
Azalide antibiotics, such as azithromycin (AZI),
are semisynthetic derivatives of ERY composed of
a 15-membered lactone ring. AZI differs from ERY
by the absence of a keto-oxygen (C9) and the addition of a methyl-nitrogen at the C10 position
(Fig. 7D). The insertion of the methyl-substituted
nitrogen in the lactone ring increases the acid stability and bioavailability compared to ERY. AZI has
been crystallized in complex with the H50S,29,68
T70S,5 and D50S69 (Table 1). Given the chemical
similarity to ERY, it is not surprising that the binding mode of AZI on the H50S and T70S is near
identical to ERY-E50S (Fig. 7E). A similar binding
site is observed on D50S; however, a slight shift is
reported in the position of the desosamine sugar as
well as a deviation in the location of the distal region
of the lactone ring (Fig. 7F).69 It is possible that the
latter deviation in the lactone results, in part, from
the unexpected finding that a second molecule of
AZI (AZI-2) interacts with this region of AZI-1 in
D50S (Fig. 7G).69 In addition, AZI-2 interacts predominantly with the tip of L22 (Fig. 7G), suggesting
that differences in the sequence of this region of L22
are responsible for influencing the binding of AZI-2
to ribosomes of different species. Kinetic and binding data also support the cooperative interaction of
two AZI molecules in D50S and a single-binding site
on E70S ribosomes.70
The 16-membered macrolides, such as tylosin
(TYL) and carbomycin (CAR), generally contain
mycaminose-mycarose disaccharides attached to
the C5 position (Fig. 8A and B). The crystal structure of TYL bound to the H50S68 reveals that despite
the larger size, the placement of the lactone ring and
C5-sugar is very similar to that observed for ERY
12
(Fig. 8C). Interestingly, TYL contains a C6-ethyl
aldehyde (Fig. 8A and B) that forms a covalent interaction with the N6 of A2602 (Fig. 8C and D). Biochemical studies have indicated that modifications
that abolish the potential of TYL to form a covalent
bond with A2062 dramatically reduce the binding
and inhibitory properties of the drug.58 CAR, and
the structurally related josamycin (JOS), bind to
the ribosome analogously as TYL and also form a
carbinolamine bond with A2062 (Fig. 8D).59,68 One
difference of CAR and JOS from TYL is the presence
of an isovalerate extension on the C5-disaccharide
(compare Fig. 8A and B). In the context of the ribosome, the C5-sugars extend from the macrolide
binding site in the tunnel back up toward the PTC,
such that the isovalerate extension of CAR/JOS overlaps the binding position of the A-tRNA (Fig. 8E).
This is consistent with the correlation between the
length of the C5-extension and the ability to inhibit
the peptidyltransferase activity, i.e., CAR completely
inhibits, TYL has a moderate effect (60%), and ERY
does not inhibit the reaction at all.71 The corollary is
that macrolides with C5-disaccharides, such as TYL,
generally permit synthesis of 2–4 amino acids before
peptidyl-tRNA drop-off occurs, whereas macrolides
with C5-monosaccharides like ERY allow synthesis
of oligopeptides of 6–8 amino acids in length.57 Curiously, the crystal structure of a small 12-membered
monosugar macrolide mythymycin (MYT) bound
to the D50S reveals that the drug does not bind in
the tunnel analogously to the other macrolides, but
rather at the PTC in the position overlapping the
A-tRNA72 (Fig. 8F). Based on its binding position,
MYT would be expected to inhibit peptide-bond
formation and prevent synthesis of oligopeptides. In
contrast, although the large polyketide compound
rapamycin (RAP) was shown to bind within the
tunnel of the D50S,73 the binding site is located adjacent to canonical macrolide binding site (Fig. 8F).
In this position, the lumen of the tunnel remains
unobstructed, explaining the lack of effect that RAP
has on translation in bacteria.73
Conclusion
The vast plethora of crystal structures of antibiotic–
ribosome complexes has strengthened our understanding of the conserved features that antibiotics
use to interact with the ribosome. At the same time,
these structures also highlight some differences that
arise due to species-specific differences as well as the
c 2011 New York Academy of Sciences.
Ann. N.Y. Acad. Sci. 1241 (2011) 1–16 Wilson
Antibiotics targeting the large ribosomal subunit
Figure 8. Binding of TYL, CAR, and JOS within the ribosomal tunnel. (A–B) Chemical structure of (A) TEL and (B) CAR. (C)
Comparison of the binding positions of ERY-E70S6 (gold) and TYL-H50S68 (cyan). (D) Comparison of the binding positions of
JOS-D50S59 (salmon) and CAR-H50S68 (cyan). (E) Comparison of ERY-E50S6 (gold), TYL-H50S68 (cyan), and JOS-D50S (salmon),
relative to A- (blue) and P-tRNA (green).74 (F) Comparison of ERY-E50S6 (gold), relative to MYT-D50S72 (green), RAP-D50S73
(cyan), ribosomal protein L22, and A-tRNA (blue).74
functional state of the ribosome—aspects that are
likely to be critical for the binding and inhibitory
activity of the antibiotics. This review provided a
structural basis for the species-specific differences
and indicated, where possible, how the nature of
the tRNA substrates and nascent chain can have a
dramatic influence on the effectiveness of antibiotic action. A challenge for the future is to better
define the physiological functional states that are
targeted by different classes of antibiotics, both biochemically and structurally. Such studies will not
only provide insight into the mechanism of action
of the antibiotics but could also provide a platform
upon which to design new improved antimicrobial
agents.
Conflicts of interest
The authors declare no conflicts of interest.
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