The molecular basis of the inhibitory activities of type A and type B synergimycins and related antibiotics on ribosomes

Synergimycins A and B act synergistically in vivo; the mixture of the two compounds is more powerful than the individual components and their combined action is irreversible. Type A (virginiamycin M, VM-like) components inactivate the donor and acceptor sites of peptidyltransferase, thus interfering with the corresponding functions of the enzyme. They block two of the peptide chain elongation steps: aminoacyl-tRNA (AA-tRNA) binding to the A site of ribosomes, and peptide bond formation with peptidyl-tRNA (pep-tRNA) at the P site. A tight (non-exchangeable) linkage of tRNA derivatives with the two ribosomal sites requires a stable interaction of their aminoacyl component with peptidyltransferase. Such interaction is prevented by VM, hence the release of AA-tRNA from the A site and of pep-tRNA from the P site upon translocation; ultracentrifugally unstable particles (60S) are thus formed. A new model for peptidyltransferase has been proposed, to account for the interference of VM with the two sites of the enzyme. The action of this antibiotic is partly due to its presence on the ribosome, and partly to the conformational alterations triggered by its binding. Type B synergimycins (VS-like) and the related 14-membered macrolides (erythromycin) have a more complex action, as revealed by copolymer-based models of cell-free protein synthesis. These antibiotics produce an inhibition of peptide bond formation, and a release of incomplete peptide chains, which processes are both template-dependent (i.e. linked to the polymerization of basic amino acids and proline). The functional interference of VS with peptidyltransferase is explained by the location of the corresponding binding site at the base of the central protuberance of 50S subunits. When ribosome.VS complexes are incubated with erythromycin, the former antibiotic is replaced by the latter; such a replacement does not occur in the presence of VM, which reduces ribosome affinity for macrolides and increases that for type B synergimycins. A study of these complex ribosomal interactions by stopped-flow spectrofluorimetry had allowed a mapping of the binding sites for the MLS antibiotics (macrolides, lincosamides, type B synergimycins) within the peptidyltransferase domain. The active component of these binding sites is represented by segments (loop V and domain II) of 23S rRNA, as indicated by protection and mutation mapping experiments, L proteins increasing the affinity of fixation and its specificity.