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

Abstract

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.

Resistance to Quinupristin-Dalfopristin Due to Mutation of L22 Ribosomal Protein inStaphylococcus aureus

“…The type B component is endowed with intrinsic fluorescence because of its picolinic moiety, and its binding to ribosomes results in a sharp increase of the fluorescence intensity proportional to the number of antibiotic-ribosome complexes formed (22). Erythromycin displaces ribosomebound type B streptogramin by competition.…”

“…Like erythromycin, streptogramin B binds stoichiometrically to the bacterial ribosome (17,37). When PI A (one of the group B molecules) binds the 50S subunits, its fluorescence increases in proportion to the concentration of the antibiotic-ribosome complex formed (22). Figure 2 shows fluorescence produced by PI A -ribosome complexes.…”

Chemical probing of virginiamycin M-promoted conformational change of the peptidyl-transferase domain

“…The relationship between several L proteins and two portions of the 23S rRNA, parts of domain V and II, with the catalytic center of this enzyme has been recognized (10)(11)(12)(13)(14). The chemical probing method devised by Noller and coworkers (15) has allowed the identification of rRNA bases involved in antibiotic binding to ribosomes: these bases are indeed protected by ribosome-bound antibiotics against the attack by nucleic acid reagents.…”

Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis

“…Pristinamycin is active against both gram-positive bacteria, especially Staphylococcus and Streptococcus spp., and some gram-negative bacteria, such as Haemophilus spp. Interestingly, both pristinamycins I and PII bind to the 50S ribosomal subunit and block protein synthesis (6,8).Although one enzyme involved in the biosynthesis of etamycin, a member of the B group of streptogramins, has been isolated recently (26), no enzymatic studies of the biosynthesis of PII A or any other member of the A group of streptogramins have been reported. However, incorporation studies of precursors labelled with radioactive and stable isotopes have established the origin of the 28 carbon atoms of PII A (16-18).…”

“…Pristinamycin is active against both gram-positive bacteria, especially Staphylococcus and Streptococcus spp., and some gram-negative bacteria, such as Haemophilus spp. Interestingly, both pristinamycins I and PII bind to the 50S ribosomal subunit and block protein synthesis (6,8).…”