Sructures of antibiotics bound to the 50S ribosomal subunit have only been reported so far (9/2003), by two groups: the research groups headed by A.Yonath and T.Steitz.
Fall 2001 we succeded to publish almost on the same day the structure of the 50S ribosomal subunit from Deinococcus radiodurans (in Cell) and structure of four complexes of the same subunit with different, clinically important antibiotics (Chloramphenicol, Clindamycin, Erythromycin, Clarythromycin und Roxythromycin). Since we were the first to publish 50S-antibiotics complexes we even got onto the cover page of Nature.
About a year later, the Yale group also published a couple of macrolide complexes with the 50S subunit from Haloarcula marismortui. The antibiotics bind in a similar position, however the orientation of the compounds (or its lactone ring) differs substantially from the one seen in Deinococcus radiodurans.
There are two plausible reasons, why the results differ: (i) H.marismortui requires high salt concentrations, which might well affect the binding of the antibiotics. (ii) A couple of rRNA bases important in the vicinity of the antibiotic binding sites deviate from those in eubacteria (H.m is an archeal organism, by that closer to eukaryots than bacterial pathogens. Therfore H. marismortui is generally resistant to a large number of different antibiotics, and to obtain quantitaive binding, high drug concentrations are usually required. D. radiodurans in contrast behaves very much like an "infectious bug" (D.r. is an eubacterial organism), so that even fractions of micro-molar concentrations are sufficient to get quantitative binding. Lucky, eh ?
More recent were the complexes with the ketolide ABT-773 and azithromycin (an azalide antibiotic also known under the trade name Zithromax). The structures are not really that new, but the publication was sitting in Structure's editorial offices for quite a while. Anyhow, azithromycin was the first macrolide, where we found two, rather than a single binding site. At that time, we still thought this to be unique, however there are more compounds behaving like azithromycin ... at least according to some recent (biochemical) data :-).
In the meantime a few more complexes with antibiotics have been published by us and the Yale group. Among those compounds, sparsomycin - a universal inhibitor like edeine - is particularly noteworthy.
An overview about various antibiotic binding sites is shown in the image(s) below. A more detailed, but still very brief description of the inhibitory mechanism of some of the compounds is given further down the page ....
The structure of the 50S subunit from Deinococcus radiodurans togehter with positions for the bound antibiotics Sparsomycin (green),Chloramphenicol (purple), Clindamycin (orange) und Erythromycin (red, standing for makrolides). The position/way of the tunnel is marked by a yellow ribbon.
was first discovered in 1947 as an antibiotic in cultures of Streptomyces venezuelae. It's a rather efficient broad spectrum antibiotic, which blocks peptide-bond formation directly. From biochemical data, eg suppression of the fragment reaction, it was well known that it should bind in the peptidyl transferase centre.
Therefore it wasn't surprising, when we located the drug right in the PTC, at the same spot, where tRNA molecules try to place their amino acids. (More surprising was the position the Yale group published recently: they located chloramphenicol down at the entrance to the tunnel, quite distant from the PTC, but that's a different story). The inhibitory mechanism in less than 10 words: In the presence of chloramphenicol, productive positioning of the tRNA becomes impossible, so that peptide bonds can not be formed. If you want a more detailed answer: see nature
Chloramphenicol has however a number of side effects, so that its usage is not that common anymore. Unfortunately, the compound can be produced at very low cost, and is hence heavily (mis)used as an additive in food-production. Shrimps or gambas (farmed crab meat) became meanwhile the major chloramphenicol consumer, which brought the drug occasionally in the headlines ... To convince yourself, try:
Clindamycin (2001)
belongs like lincomycin to the class of lincosamides. The lincosamides share an undesired property with the macrolides and streptogramins B: a single rRNA base modification (at A2058 in E.coli) - either through methylation or mutation - is sufficient to render the ribosome resistant against the three classes of antibiotics (named MLSB resistance).
Clindamycin binds like chloramphenicol in the active site of the 50S subunit. The binding sites of chloramphenicol and clindamycin are actually overlapping (i.e. the proline moiety occupies the same place as chloramphenicols phenyl moeity), a good reason not the combine the two drugs during medication. However, there is a subtle difference in the inhibitory activity: chloramphenicol blocks the A-site, to which the new amino acid is delivered. Clindamycin in contrast blocks both the A- and the P-site (roughly speaking the location of the C-terminal of the nascent chain).
Sparsomycin (2002/3)
is like edeine or pactamycin (or puromycin) a universal antibiotic, which inhibits protein synthesis in all organisms. That's not exactly a desired property of a good drug. Sparsomycin is therefore not used as an anti-infective, but rather as a anti-tumor drug. Besides, its an excellent tool to study different stages of protein synthesis.
The inhibitory activity of this compound is - despite the structural studies - still a bit puzzling.
The position of sparsomycin in the 50S subunit has been deduced by both Yonaths/Fucinis and Steitz' group. Both found sparsomycin stacking onto the same rRNA base, but on opposite sides of the base. Both results imply, that sparsomycins primary action relies on the interaction with the P-site tRNA. Thats at least in agreement with earlier biochemical experiments showing that sparsomycin enhances the affinity of tRNA for the P-site (i.e., the tRNA remains stuck in the P-site, which blocks translocation and therefore also protein synthesis).
Makrolides (2001)
Erythromycin
Clarithromycin
Roxithromycin
The macrolides are the broadest and probably most interesting class of compounds studied so far. macrolides are generally composed of a lactone ring (roughly 8-20 membered), a desosamine sugar and varying decorations.
The classic macrolides like erythromycin posses a cladinose sugar, which was originally believed to be indispensible. More recent semi-synthetic derivatives can composate for the cladinose by addition of other functional moieties, like keto- or azo-groups.
The mechanism of the inhibition by macrolides is most beautifully (:-)) captured in this image:
macrolides block the entrance to the ribosomal exit tunnel, without blocking the PTC of the 50S subunit (there are exceptions though). The ribosome can still produce a short peptide chain, before the traffic jams. Those who tried to cross the Hamburger Elbtunnel recently, know how efficient such a mechanism is. Anyhow, the ribosome is stalled, which stops the growth of the organism.
In contrast to cell wall antibiotics like penicillin, the macrolides act only bacteriostatic.
Makrolides 2002 (Carbomycin A, Tylosin .... )
In the meantime, also the Yale group published a couple of macrolide-50S complexes, again using Haloarcula marismortui ribosomes, which are unfortunately (:-)) somewhat resistant against most of these compounds. Anyhow, the complex with carbomycin A ist still quite remarkable: it seems, that carbomycin A binds covalently to the ribosomal, which implies a stronger binding to the 50S subunit, considerably stronger than erythromycin which exhibits already a high affinity.
Ketolides (2003):
ABT-773 ... meanwhile also known as Cethromycin, was the first ketolide we could bind to (and locate in) our crystals of the 50S subunit. ABT-773 is one of the compounds without a cladinose sugar, which is replaced by a keto. In addition, ABT-773 carries a carbamate group and a quinolylallyl, which both contribute to the binding. The quinolylallyl reaches domain II (see 50S structure), a common way of macrolide derivatives to overcome typical resistances (as long as they are based on target modification).
The images show the location of ABT-773 within the tunnel (right) and a roughly 35A deep section of the tunnel around ABT-773 (left).
Unfortunately time is too short for a more detailed description, but check the List of puclications. We will also try to make the PDF's available ... but that takes time ... (copyright issues).
One of the most recent macrolides developed is the ketolide Telithromycin (Ketek®), which has been approved July 2001 in Europe. It exhibits a superior antimicrobial activity against a wide spectrum of pathogens.
Telithromycin is rather similar to ABT-773 (but a much better drug), also possesing a cyclic carbamate group (red) attached to lactone ring (black), the keto (pink), no cladinose and a quninolylallyl moiety (blue), which is in contrast to ABT-773 attached to the carbamate group.
Left image: telithromycin binds - unexpectedly (?) - slightly different than ABT-773, and some of the nucleotides involved in binding differ. It seems that the position of the quinolylallyl makes the difference.
Right image: the quinolylallyl of telithromycin interacts with four nucleotides of domain II of 23S rRNA. The difference in the interactions with domain II lead to a slighly different orientation of the drug compared to ABT-773 at the entrance of the tunnel.
The inhibitory mechanism - blockage of the path of the nascent chain - remains however the same.
Azithromycin (Zithromax), is a rather normal macrolide with a desosamine and a cladinose sugar. However, the insertion of the azo-group into the lactone ring, which therefore becomes 15 membered. The azo-group leads to a better acid stability and bio-availibility. For example, its sufficient to apply a single dose of azithromycin per day, compared to the common 3 doses per day. Its also special, since its the first macrolide possesing two binding sites on the 50S subunit. The image on the left shows a section through the ribosome with the tunnel highlighted in gray. The two ribosomal proteins L4 (green) and L22(blue), which are involved in the binding of the secondary azihtromycin molecule, are also shown.
Interestingly, H.marismortui 50S subunits contain only the primary azihtromycin site, with an, as usual, different oriented lactone ring..
The image on the right highlights the positions of both azithromycin molecules withing the tunnel. The azithromycin molecules interact directly with each other, mediated through the azo-group, which is hence particularly important for this special arrangement.
Makrolides (2003): Troleandomycin
is a three fold acetylated variant of oleadormycin, where it derives its name from. The acetlyation modifies many of the interactions with 23S rRNA. It's surprising enough that troleandomycin binds at all. And troleandomycin sits only slightly deeper inside the tunnel. However, most remarkable is the conformational change induced by the compound.
In the native structure, L22 delineates the tunnel wall, but upon binding of troleandomycin, the hairpin loop of L22 flips towards the other side of the tunnel. In this conformation, L22 restricts the passage through the tunnel, such that bulky side chains won't be able to pass.
This result is the first direct observation of a functionally important conformational transition inside the tunnel.
L22(nativ), Troleandomycin
The unusual side effect of troleandomycin correlates with another, independent observation. The production of the Secretory Monitoring Protein (SecM) is accompanied by a transient pause of protein synthesis. A specific sequence in the nascent chain, which still resides within the tunnel, seems to stall the ribosome, at least for a short while. This effect is independent of the sequence context, and only happens if a hydrophobic stretch of the nascent chain is in the vicinity of L22. The natural conclusion is, that some elements within the tunnel can recognize specific sequences, and regulate their production.
L22(flip), Troleandomycin
This mechanism can be abolished by certain mutation in the loop of L22, just in the region which is essential for the flip of its hairpin loop. It's therefore plausible to assume, that the same conformational transition we saw in the presence of troleandomycin is also responsible for sequence specific stalling of the ribosome. However, the ultimative proof is still lacking ...
The proposed movement of the hairpin loop of L22 induced by troleandomycin is shown as a small (170kB) and simple animation.
Closely related to the activity of antibiotics is the loss of power due to emerging drug resistances, which became a major health problem. A few bugs became resistant against essentially ALL therapeutic drugs, which is extremly for elderly patients, or patients with a compromised immune response. We collected a bit of information about antibiotics resistences on a separate page.
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