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J. Biol. Chem., Vol. 281, Issue 32, 23103-23110, August 11, 2006

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Unraveling New Features of Clindamycin Interaction with Functional Ribosomes and Dependence of the Drug Potency on Polyamines^*

From the Laboratory of Biochemistry, School of Medicine, University of Patras, 26500 Patras, Greece

The effect of spermine on the inhibition of peptide-bond formation by clindamycin, an antibiotic of the Macrolide-Lincosamide-Streptogramins_B 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 °C as opposed to 5 °C). If this is not met, the inhibition is simple competitive. It was found that at 25 °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 °C nor can they stimulate the clindamycin binding to ribosomes. Our kinetic results correlate well with photoaffinity labeling data, suggesting that at 25 °C polyamines bound at the vicinity of the drug binding pocket affect the tertiary structure of ribosomes and influence their interaction with clindamycin.

Received for publication, April 5, 2006 , and in revised form, June 6, 2006.

^* This work was supported by University of Patras (Programme K. Karatheodori) Research Committee Grant B115. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental material.

¹ To whom correspondence should be addressed. Tel.: 302610-996124; Fax: 302610-997690; E-mail: dimkal{at}med.upatras.gr.

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