Localization of virginiamycin S binding site on bacterial ribosome by fluorescence energy transfer

Abstract

Virginiamycin S, a type B synergimycin inhibiting protein synthesis in bacteria, competes with erythromycin for binding to the 50S ribosomal subunits; the mechanism of action of the two antibiotics is unclear. Energy-transfer experiments between virginiamycin S (which is endowed with inherent fluorescence due to its hydroxypicolinyl moiety) and fluorescent coumarinyl derivatives of ribosomal proteins L7 and L10 have been carried out to locate the binding site of this antibiotic on the ribosome. Previous studies have indicated that two L7/L12 dimers can attach respectively to a strong binding site located on the central protuberance and to a weak binding site located on the stalk of the 50S subunits and that protein L10 is located at the base of the stalk. The distance between ribosome-bound virginiamycin S and a fluorophore located at the strong binding site of proteins L7/L12 (Lys-51 of L7) was found to be 56 (+/- 15) A. Virginiamycin S, on the other hand, was located at a distance exceeding 67 A from the weak binding site of L7/L12 dimers. A fluorophore positioned on the unique cysteine (Cys-70) of protein L10 and ribosome-bound virginiamycin S proved to be more than 60 A apart. From data available on the location of proteins L7/L12 and L10, a model is proposed, whereby the virginiamycin S binding site is placed at the base of the central protuberance of the 50S subunits, in proximity of the presumptive peptidyl transferase center. The binding sites of macrolides and lincosamides (related antibiotics of the MLS group) are expected to be very close to that of virginiamycin S.

Affinity labeling of the virginiamycin S binding site on the bacterial ribosome

“…In agreement with such a conclusion are previous studies indicating the overlapping of virginiamycin S binding sites and erythromycin-binding sites within the peptidyltransferase domain (Di Giambattista et al, 1987). The former binding site has been recently located, by nonradiant energy transfer, at the base of the central protuberance (Di Giambattista et al, 1986), the presumptive location of the peptidyltransferase center. To this region, most of the above-mentioned proteins (L2, L15, L16, L18, L22, L27) have been assigned by immune electron microscopy and cross-link experiments (Stoffler & Stoffler-Meilicke, 1986;Redl et al, 1989).…”

“…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 A-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.…”

“…The location of the virginiamycin S binding site on bacterial ribosomes has been established by nonradiant energy transfer between ribosome-bound virginiamycin S (endowed with inherent fluorescence) and fluorescent coumarinyl residues grafted onto L7, L10, and L12 proteins of known topological situation. By this work, a region at the base of central protuberance, the putative peptidyltransferase domain, has been identified (Di Giambattista et al, 1986). This observation, in addition to the virginiamycin S promoted inhibition of peptide bond formation (Chinali et al, 1988a,b), has suggested the possibility of labeling the peptidyltransferase domain by use of an affinity-labeling derivative of virginiamycin S. The main difficulties for this kind of approach are as follows: (a)…”

Les antibiotiques inhibiteurs de la synthèse protéique

“…De plus, ces antibiotiques stimulent le relâchement des chaînes peptidi ques en formation au niveau d'acides aminés basiques [23]. Le site d'atta chement des synergimycines B a été localisé à la base de la protubérance centrale, l'emplacement présumé de la peptidyl-transférase [40]. Cette observation a été confirmée par les résultats d'expériences de protection exercée par la vernamycine B sur les bases A752, A2062 et G2505 de l'ARNt 23S vis-à-vis de l'action de divers agents chimiques [22].…”