PMID:16760473
| Citation |
Kouvela, EC, Petropoulos, AD and Kalpaxis, DL (2006) Unraveling new features of clindamycin interaction with functional ribosomes and dependence of the drug potency on polyamines. J. Biol. Chem. 281:23103-10 |
|---|---|
| Abstract |
The effect of spermine on the inhibition of peptide-bond formation by clindamycin, an antibiotic of the Macrolide-Lincosamide-StreptograminsB family, was investigated in a cell-free system derived from Escherichia coli. In this system peptide bond is formed between puromycin, a pseudo-substrate of the A-site, and acetylphenylalanyl-tRNA, bound at the P-site of poly(U)-programmed 70 S ribosomes. Biphasic kinetics revealed that one molecule of clindamycin, after a transient interference with the A-site of ribosomes, is slowly accommodated near the P-site and perturbs the 70 S/acetylphenylalanyl-tRNA complex so that a peptide bond is still formed but with a lower velocity compared with that observed in the absence of the drug. The above mechanism requires a high temperature (25 degrees C as opposed to 5 degrees C). If this is not met, the inhibition is simple competitive. It was found that at 25 degrees C spermine favors the clindamycin binding to ribosomes; the affinity of clindamycin for the A-site becomes 5 times higher, whereas the overall inhibition constant undergoes a 3-fold decrease. Similar results were obtained when ribosomes labeled with N1-azidobenzamidinospermine, a photo-reactive analogue of spermine, were used or when a mixture of spermine and spermidine was added in the reaction mixture instead of spermine alone. Polyamines cannot compensate for the loss of biphasic kinetics at 5 degrees C nor can they stimulate the clindamycin binding to ribosomes. Our kinetic results correlate well with photoaffinity labeling data, suggesting that at 25 degrees C polyamines bound at the vicinity of the drug binding pocket affect the tertiary structure of ribosomes and influence their interaction with clindamycin. |
| Links |
PubMed Online version:10.1074/jbc.M603263200 |
| Keywords |
Anti-Bacterial Agents/chemistry; Azides/chemistry; Clindamycin/chemistry; Drug Interactions; Escherichia coli/metabolism; Kinetics; Models, Chemical; Models, Molecular; Peptides/chemistry; Polyamines/chemistry; Protein Structure, Tertiary; RNA, Ribosomal, 23S/chemistry; Ribosomes/chemistry; Ribosomes/metabolism; Spermine/analogs & derivatives; Spermine/chemistry |
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