Effects of Two Photoreactive Spermine Analogues on Peptide Bond Formation and Their Application for Labeling Proteins in <i>Escherichia coli</i> Functional Ribosomal Complexes

Xaplanteri

Dinos

et al. 2004

Self Cite

The effects of spermine on peptidyltransferase inhibition by an aminohexosylcytosine nucleoside, blasticidin S, and by a macrolide, spiramycin, were investigated in a model system derived from Escherichia coli, in which a peptide bond is formed between puromycin and AcPhe-tRNA bound at the P-site of poly(U)-programmed ribosomes. Kinetics revealed that blasticidin S, after a transient phase of interference with the A-site, is slowly accommodated near to the P-site so that peptide bond is still formed but with a lower catalytic rate constant. At high concentrations of blasticidin S (>10 ؋ K i ), a second drug molecule binds to a weaker binding site on ribosomes, and this may account for the onset of a subsequent mixed-noncompetitive inhibition phase. Spermine enhances the blasticidin S inhibitory effect by facilitating the drug accommodation to both sites. On the other hand, spiramycin (A) was found competing with puromycin for the A-site of AcPhe-tRNA⅐poly(U)⅐70 S ribosomal complex (C) via a two-step mechanism, according to which the fast formation of the encounter complex CA is followed by a slow isomerization to a tighter complex, termed C*A. In contrast to that observed with blasticidin S, spermine reduced spiramycin potency by decreasing the formation and stability of complex C*A. Polyamine effects on drug binding were more pronounced when a mixture of spermine and spermidine was used, instead of spermine alone. Our kinetic results correlate well with cross-linking and crystallographic data and suggest that polyamines bound at the vicinity of the antibiotic binding pockets modulate diversely the interaction of these drugs with ribosomes.Blasticidin S and spiramycin are representatives of two distinct antibiotic families, the aminohexosylcytosine nucleosides and the 16-membered lactone ring macrolides, respectively, both of which have been proposed to inhibit protein synthesis by binding to the large ribosomal subunit. Blasticidin S consists of a cytosine bonded to a pyranose ring, to which an amino acid-like substituent is attached (see Fig. 1). Therefore, it is not surprising that blasticidin S and puromycin have been considered as iso-structural, both with one another and with the 3Ј-end of aminoacyl-tRNA (1, 2). Consistently, blasticidin S has been found to inhibit the binding of CACCA (Phe) to the A-site of ribosomes (3) and to compete with designated A-site inhibitors (4). In addition, the inhibition of peptidyl-or AcPhe 1 -puromycin synthesis by this antibiotic shows a competitive phase in concert with noncompetitive or mixed-noncompetitive phases (5, 6), a fact supporting the notion that blasticidin S influences, at least transiently, the affinity of the A-site. Further support derives from chemical probing studies in Escherichia coli ribosomes (7), suggesting that blasticidin S protects A2439, one of the three nucleosides in domain V of 23 S rRNA, which show altered chemical reactivity on removal of the aminoacyl group from A-site-bound tRNAs (8). Moreover, A2439 is one of the preferable cross-linking...

Section: Polyamines Reduce the Binding Of Spiramycin To Complex C-earmentioning

confidence: 70%

Polyamines Affect Diversely the Antibiotic Potency

Xaplanteri

Dinos

et al. 2004

Self Cite

“…The photoreactive analogue retains a charge in the vicinity of the nearest amino group, closely resembling spermine. This may explain the fact that ABA-spermine in the dark displays similar biological activity to that determined for spermine when used as a component of the ionic environment of ribosomes (27). Photolabeling of ribosomes with ABA-spermine under mild irradiation conditions results in covalent and specific binding of spermine with ribosomal proteins (27) and various groups in rRNA (28).…”

Section: Aba-spermine Cross-linking In 23 S Rrna Interferes With Clinmentioning

confidence: 78%

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

Kouvela

Kalpaxis

2006

Self Cite

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 N 1 -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.Lincomycin and clindamycin ( Fig. 1) are lincosamides, still used as therapeutic agents in human diseases and some animal infections. Clindamycin, a semisynthetic derivative of lincomycin (7(S)-chloro-7-deoxylincomycin), is usually more active than the parent compound in the treatment of bacterial infections, in particular those caused by anaerobic species (1).Lincosamides act on the large ribosomal subunit and share an overlapping binding site with macrolides, streptogramins B, chloramphenicol, and other antibiotics affecting the PTase 2 center, as deduced from competition experiments and crossresistance data (for review, see Ref.2) as well as from twodimensional transferred nuclear Overhauser effect spectroscopy analysis (3). In agreement with most of these observations, chemical footprinting analysis (4, 5) and crystallographic studies (6, 7) have revealed that lincosamides interact with both the A-site and the P-site on the 50 S ribosomal subunit and would be expected to hamper the positioning of aminoacyl moieties of tRNAs at both sites. This last view is consistent with earlier binding studies investigating the competition for binding to ribosomes between lincomycin and 3Ј-terminus pentanucleotide fragments from N-AcLeu-tRNA Leu , Leu-tRNA Leu , and Phe-tRNA Phe (8, 9) as well as with the finding that lincosamides stimulate oligopeptidy...

“…Our results are discussed in view of NMR and crystallographic studies on the interactions of these drugs with the ribosome. 4 Cl) 70 S ribosomes and partially purified translation factors were prepared from E. coli K12 cells as described elsewhere (29). E. coli TA531 cells that lack chromosomal rrn alleles but contain wild-type or mutant pKK3535 plasmids expressing wild-type or mutated 23 S rRNA (U2609C or U754A), respectively, were kindly provided by Prof. A. S. Mankin (University of Illinois) and used as a source of ribosomes in studying the effect of certain mutations on the mechanism of drug binding to E. coli ribosomes.…”

mentioning

confidence: 99%

Stepwise Binding of Tylosin and Erythromycin to Escherichia coli Ribosomes, Characterized by Kinetic and Footprinting Analysis

Kouvela

Dinos

et al. 2008

Self Cite

Erythromycin and tylosin are 14-and 16-membered lactone ring macrolides, respectively. The current work shows by means of kinetic and chemical footprinting analysis that both antibiotics bind to Escherichia coli ribosomes in a two-step process. The first step established rapidly, involves a low-affinity binding site placed at the entrance of the exit tunnel in the large ribosomal subunit, where macrolides bind primarily through their hydrophobic portions. Subsequently, slow conformational changes mediated by the antibiotic hydrophilic portion push the drugs deeper into the tunnel, in a high-affinity site. Compared with erythromycin, tylosin shifts to the high-affinity site more rapidly, due to the interaction of the mycinose sugar of the drug with the loop of H35 in domain II of 23 S rRNA. Consistently, mutations of nucleosides U2609 and U754 implicated in the high-affinity site reduce the shift of tylosin to this site and destabilize, respectively, the final drug-ribosome complex. The weak interaction between tylosin and the ribosome is Mg 2؉ independent, unlike the tight binding. In contrast, both interactions between erythromycin and the ribosome are reduced by increasing concentrations of Mg 2؉ ions. Polyamines attenuate erythromycin affinity for the ribosome at both sequential steps of binding. In contrast, polyamines facilitate the initial binding of tylosin, but exert a detrimental, more pronounced, effect on the drug accommodation at its final position. Our results emphasize the role of the particular interactions that side chains of tylosin and erythromycin establish with 23 S rRNA, which govern the exact binding process of each drug and its response to the ionic environment.Erythromycin and tylosin (Fig.