The streptogramins and related antibiotics (the lincosamides and macrolides) (MLS) are important inhibitors of bacterial protein synthesis. The key reaction in this process is the formation of a peptide bond between the growing peptide chain (peptidyl-tRNA) linked to the P-site of the 50S ribosome and aminoacyl-tRNA linked to the A site. This reaction is catalysed by the peptidyl transferase catalytic centre of the 50S ribosome. Type A and B streptogramins in particular have been shown to block this reaction through the inhibition of substrate attachment to the A and P sites and inhibition of peptide chain elongation. Synergy between type A and B components results from conformational changes imposed upon the peptidyl transferase centre by type A compounds and by inhibition of both early and late stages of protein synthesis. The conformational change increases ribosomal affinity for type B streptogramins. Microbial resistance to the MLSB antibiotics is largely attributable to mutations of rRNA bases, producing conformational changes in the peptidyl transferase centre. This can result in resistance to a single inhibitor or to a group of antibiotics (MLSB). The activity of type A streptogramin is retained thus explaining the improved inhibitory action of the combined streptogramins against macrolide and lincosamide-resistant strains. However, the development of resistance to the streptogramins may be less of a problem because of the synergic effect of type A and B compounds which has also been demonstrated in strains resistant to MLSB i.e., high level resistance to the combined streptogramins is only likely when type A streptogramin resistance determinants are present along with type B streptogramin resistance determinants.

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.

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Virginiamycin S (VS, a type B synergimycin) inhibits peptide bond synthesis in vitro and in vivo. The attachment of virginiamycin S to the large ribosomal subunit (50S) is competitively inhibited by erythromycin (Ery, a macrolide) and enhanced by virginiamycin M (VM, a type A synergimycin). We have previously shown, by fluorescence energy transfer measurements, that virginiamycin S binds at the base of the central protuberance of 50S, the putative location of peptidyltransferase domain [Di Giambattista et al. (1986) Biochemistry 25, 3540-3547]. In the present work, the ribosomal protein components at the virginiamycin S binding site were affinity labeled by the N-hydroxysuccinimide ester derivative (HSE) of this antibiotic. Evidence has been provided for (a) the association constant of HSE-ribosome complex formation being similar to that of native virginiamycin S, (b) HSE binding to ribosomes being antagonized by erythromycin and enhanced by virginiamycin M, and (c) a specific linkage of HSE with a single region of 50S, with virtually no fixation to 30S. After dissociation of covalent ribosome-HSE complexes, the resulting ribosomal proteins have been fractionated by electrophoresis and blotted to nitrocellulose, and the HSE-binding proteins have been detected by an immunoenzymometric procedure. More than 80% of label was present within a double spot corresponding to proteins L18 and L22, whose Rfs were modified by the affinity-labeling reagent. It is concluded that these proteins are components of the peptidyltransferase domain of bacterial ribosomes, for which a topographical model, including the available literature data, is proposed.