Interaction between virginiamycin S and ribosomes is partly provided by a salt bridge with a magnesium ion

Abstract

Type B streptogramins, such as virginiamycin S (VS), are cyclic hexadepsipeptides, inhibiting protein synthesis in prokaryotes. L-Thr connects a 3-hydroxypicolinyl residue (3-OH-Pic) to the peptide lactone ring. The fluorescence intensity of 3-OH-Pic is strongly increased by chelation to alkaline earth cations or binding to ribosomes. Similar behavior of the ribosome-VS complex and the VS-Mg chelate provides strong evidence for the presence of a VS-Mg chelate within the ribosomal binding site. Different models involving the ribosome binding of either members of the VS-Mg2+ chelate or both have been tested by fluorescence lifetime measurements, equilibrium titrations, and stopped-flow spectrofluorometry. Our data strongly suggest that (a) the interaction between VS and the ribosome is partly provided by a salt bridge between suitable acceptor atoms of the ribosome and the 3-OH-Pic residue, (b) Mg2+ can be exchanged by Mn2+ without dissociation of the ribosome-VS complex, (c) Mg2+ coordinates to the negative form of the 3-OH-Pic residue, probably via an interaction with the phenolate oxygen and the amide carboxyl group, and (d) the picolinyl residue is essential for the biological activity, as indicated by the lack of activity when the latter is replaced by a serine derivative.

Structural basis for streptogramin B resistance in Staphylococcus aureus by virginiamycin B lyase

“…In retrospect, the similarities in Mg 2ϩ coordination are not surprising as type B streptogramins can chelate divalent metal ions. Also, the Mg 2ϩ mode of binding involving the 3-hydroxypicolinic acid moiety had been correctly predicted based on spectroscopic analyses more than a decade ago (21). However, what is surprising is that the intrinsic chelating properties of type B streptogramins make these antibiotics inherently susceptible to enzymatic degradation as the metal ion weakens the threonyl C ␣ -H bond.…”

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

“…Post-translocation complex of poly(U)-programmed ribosomes, complex C, carrying tRNA Phe and Ac[ 3 H]Phe-tRNA at the E-and P-sites, respectively, was prepared in buffer A (100 mM Tris/HCl, pH 7.2, 4.5 mM Mg(CH 3 COO) 2 , 150 mM NH 4 Cl, and 6 mM 2-mercaptoethanol) and purified according to Dinos et al (31). Whenever required, 100 M spermine or a mixture of 50 M spermine and 2 mM spermidine was also included in buffer A.…”

“…Binding of Antibiotics and Probing of Complexes-Complex C at 100 nM was incubated alone or with antibiotics (I) at concentration equal to 50 ϫ K i in 100 l of buffer B (HEPES-KOH, pH 7.2, 4.5 mM Mg(CH 3 COO) 2 , 150 mM NH 4 Cl, 5 mM dithiothreitol) at 25°C, either for 10 s or for 8 ϫ t1 ⁄ 2 min, dependent on whether the encounter complex CI or the final complex C*I was desired to be probed, respectively. The term t1 ⁄2 , which represents the half-life for the attainment of equilibrium between complex C and the drug, was calculated through the relationship,…”

“…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.…”

“…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. Prior to their use, ribosomes were activated in buffer containing 20 mM Mg(CH 3 COO) 2 and 150 mM NH 4 Cl by incubation for 20 min at 42°C. Samples were then cooled at 0°C and the Mg 2ϩ concentration was normalized to 4.5 mM.…”