Molecular Microbiology: F. Schlünzen, Pyetan, P. Fucini, A. Yonath and J.M. Harms, AInhibition of Peptide Bond Formation by Pleuromutilins: the Structure of the 50S Ribosomal Subunit from Deinococcus radiodurans in Complex with Tiamulin. Molecular Microbiology (2004) see list of publications
Tiamulin is a potent inhibitor of protein synthesis in bacteria. This prominent member of the pleuromutilin class of antibiotics is soluble in water and readily absorbed. Therefore it can be easily administered to animals (main goal: treat swine dysentery).
However, until now no pleuromutilin derivative made it to human use, but the increasing number of pathogens resistant to common antibiotics has raised a new interest in pleuromutilin derivatives for human therapy (Bacque et al., 2002, 2003; Brooks et al., 2001; Pearson et al., 2002; Springer et al., 2003).
Following many other classes of antibiotics, we managed to shed light on the binding mode and the mode of action of tiamulin on the large ribosomal subunit (50S) from Deinococcus radiodurans.
Right: Tiamulin is located within the peptidyl transferase center (PTC) of the 50S ribosomal subunit. Its tricyclic mutilin core is positioned in a tight pocket at the A-tRNA binding site (built by G2061, A2451, C2452, A2503, U2504, G2505, U2506 -- E.coli numbering).
Its extension, which protrudes from the C12 of its mutilin core to the other side of the ribosomal tunnel (entrance), partially overlaps with the P-tRNA binding site.
Blue: Hydrophobic binding partners of the 23S rRNA
Purple: Binding partners for hydrogen bonds
Thus, tiamulin directly inhibits peptide bond formation (in agreement with early biochemical findings[Drews et al., 1975; Egger and Reinshagen, 1976a; Hodgin and Hogenauer, 1974; Hogenauer, 1975]). tRNA may still bind in A- or P-site but only in 'none-productive' conformations.
For ribosomes which already have bound a tRNA on A- or P-site (or both) parts of the binding site for tiamulin are occupied, thus the drug won't bind. This agrees with another early finding (by Dornhelm and Hogenauer, 1978) which showed that ribosomes already engaged in the process of elongation are not influenced by pleuromutilin drugs.
Right: Overlap of the binding sites for tiamulin and the A- and P-tRNA ends (orange: tiamulin; blue: A-CCPuro; xxxx: P-CCPuro)
Tiamulin: Binding to the 23S rRNA
Tiamulin exclusively binds to domain V of the 23S RNA through an extensive network of hydrophobic interactions (namely G2061, A2062, C2063, A2451, C2452, A2503, U2504, G2505, U2506, U2585 and C2586 ) and with hydrogen bonds to G2061 and U2585 (numbering E.coli ).
Up:
Tiamulin (Egger and Reinshagen, 1976b), the derivative of the natural antibiotic pleuromutilin (Kavanagh et al., 1951), consists of a tricyclic nucleus composed of a cyclo-pentanone, cyclo-hexyl and cyclo-octane, and a (((2-(diethylamino)ethyl)thio)-acetic acid) side-chain on C14 of the octane ring. This side-chain differs for all antibiotics of the pleuromutilin group.
Hydrogen bonds are possible between the hydroxyl-group on C11 of the octane-ring and 23S RNA, from the side-chain the only keto-group and the sulfur atom are in hydrogen bond distance to RNA.
The natural pleuromutilin is a product of Pleurotus mutilus (nowadays named Clitopilus scyphoides).
Right: Clitopilus scyphoides, an eatable mushroom.
(Foto from Markus Wilhelm. www.pilze-basel.ch )
Right:
Overview of the 23S binding partners of tiamulin. In addition biochemical findings are marked.
Figure above:
Structure of tiamulin with its binding partners
(A) View of the local environment of tiamulin (orange) in a orientation rotated by 10 degrees from the view shown in figure 2D. Nucleotides involved in hydrogen bonds and hydrophobic interactions are colored in silver-blue and white, respectively. Hydrogen bonds are shown as dotted green lines. For sake of clarity nucleotides not involved in binding have been omitted. (B) An orientation 90 degree rotated to (A).
Comparison of binding site from ribosomal antibiotics (on 50S)
Right:
The tiamulin binding site overlaps with A- und P-tRNA and several antibiotic binding sites.
Orange: Tiamulin
Blue: A-CCPuro
xxxx: P-CCPuro
Yellow: Dalfopristin (Streptogramin A)
Purple: Chloramphenicol
Light grey: Clindamycin
Green: Quinopristin (Streptogramin B)
Red: Erythromycin (Makrolide)
From: [Bashan et al., 2003; Harms et al., 2004; Schlunzen et al., 2001; Schlunzen et al., 2003]
~90o rotated
Interestingly the right image shows how close the binding sites of the antibiotics really are. This makes multi-resistance mutations in the 23S RNA easy understandable.
Down: Although dalfopristin (streptogramin A) and tiamulin share most of the 23S RNA binding partners, the long common part of the sidechain (-CH2-CH2-CH-N-(CH2-CH3)2), shows and thus binds in totally different directions and environments.
Comparison of the 23S RNA environment of this two antibiotics delivers only 2 significant differences: Nucleotide U2585, which has an orientation ~180o rotated from the native (similar tiamulin) conformation. And A2062, which needs to have a distinct orientation to allow the synergistic binding of both streptogramins (A and B) together (see our page about streptogramins).
This common binding site raised the idea to try binding of tiamulin and streptogramin B (mikamycin B) together, with hope for enhancement of the streptogramin B binding. Well, unfortunately it didn't work, tiamulin could be clearly detected but not a piece of mikamycin. Nevertheless, this can be counted as an additional proof for the need of the distinct orientation of A2602.
Overlapping binding sites and possible hybrid-antibiotics
Comparing the overlapping binding sites suggests modifications, which could result in the enhancement of antimicrobial activity of new derivatives of known antibiotics.
Right:
The methyl group of the (((2-(diethylamino)ethyl)thio)-acetic acid) side-chain of tiamulin is within binding distance to the ethyl group on the macrocyclic ring of quinupristin. Though attachment of the thio-acetic moiety to streptogramins B would prevent synergistic binding, it might still substantially enhance streptogramin B binding. Such a modified streptogramin would not only block the entrance to the ribosomal exit tunnel, but also affect peptide bond formation directly. Down, right:
It also might be possible to combine clindamycin and pleuromutilins in such a way, that the sugar-moiety of clindamycin would serve as a potential C12-extension of the pleuromutilins cyclo-octane. Down, left:
Finally, the end of chloramphenicol may help building an extension on the C6-mathyl of the cyclo-hexane.
Tiamulin und Quinopristin
Tiamulin und Chloramphenicol
Tiamulin und Clindamycin
Well, actually these are only raw ideas derived f rom our (two physicists) observations and findings of antibiotic binding sites. The positional eror from the limited resolution of 3-3.5A should not be forgotten in further developements. Nevertheless, several antibiotic-ribosome complexes show, that the binding environment 23S RNA is quite flexible.
Our result shed a lot of light on the binding mode of tiamulin and the pleutomutilin group of antibiotics and will hopefully be a good help in development of new antibiotics or pleuromutilin-derivatives also for human use.
2004
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