RESUMEN
Viomycin is a tuberactinomycin antibiotic essential for treating multidrug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of â¼ 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Factor G de Elongación Peptídica/antagonistas & inhibidores , Biosíntesis de Proteínas/efectos de los fármacos , Viomicina/farmacología , Bacterias/metabolismo , Relación Dosis-Respuesta a Droga , Guanosina Trifosfato/química , Probabilidad , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Viomicina/metabolismoRESUMEN
Capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics which are particularly active against Mycobacterium tuberculosis, including multidrug resistant strains. Both antibiotics bind across the ribosomal interface involving 23S rRNA helix 69 (H69) and 16S rRNA helix 44 (h44). The binding site of tuberactinomycins in h44 partially overlaps with that of aminoglycosides, and they share with these drugs the side effect of irreversible hearing loss. Here we studied the drug target interaction on ribosomes modified by site-directed mutagenesis. We identified rRNA residues in h44 as the main determinants of phylogenetic selectivity, predict compensatory evolution to impact future resistance development, and propose mechanisms involved in tuberactinomycin ototoxicity, which may enable the development of improved, less-toxic derivatives.
Asunto(s)
Antituberculosos/farmacología , Capreomicina/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Ribosomas/efectos de los fármacos , Viomicina/farmacología , Aminoglicósidos/farmacología , Antituberculosos/metabolismo , Antituberculosos/toxicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Capreomicina/metabolismo , Capreomicina/toxicidad , Farmacorresistencia Bacteriana Múltiple/genética , Enviomicina/análogos & derivados , Enviomicina/farmacología , Enviomicina/toxicidad , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , ARN Ribosómico 16S/metabolismo , ARN Ribosómico 23S/metabolismo , Viomicina/metabolismo , Viomicina/toxicidadAsunto(s)
Antituberculosos/metabolismo , Proteínas Bacterianas/química , Capreomicina/metabolismo , Ingeniería Metabólica/métodos , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos/genética , Péptido Sintasas/química , Proteínas Recombinantes/química , Viomicina/metabolismo , Antituberculosos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Capreomicina/química , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Escherichia coli/enzimología , Escherichia coli/genética , Tuberculosis Extensivamente Resistente a Drogas/tratamiento farmacológico , Tuberculosis Extensivamente Resistente a Drogas/microbiología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Plásmidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Streptomyces lividans/enzimología , Streptomyces lividans/genética , Homología Estructural de Proteína , Transformación Genética , Viomicina/químicaRESUMEN
Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-A resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in the pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.
Asunto(s)
Capreomicina/química , Capreomicina/metabolismo , Ribosomas/metabolismo , Thermus thermophilus/metabolismo , Viomicina/química , Viomicina/metabolismo , Antituberculosos/química , Antituberculosos/metabolismo , Cristalografía por Rayos X , Datos de Secuencia Molecular , Estructura Molecular , Unión Proteica , Estructura Secundaria de Proteína , ARN de Transferencia/genética , Ribosomas/química , Ribosomas/genéticaRESUMEN
BACKGROUND: The peptide antibiotic viomycin inhibits ribosomal protein synthesis, group I intron self-splicing and self-cleavage of the human hepatitis delta virus ribozyme. To understand the molecular basis of RNA binding and recognition by viomycin, we isolated a variety of novel viomycin-binding RNA molecules using in vitro selection. RESULTS: More than 90% of the selected RNA molecules shared one continuous highly conserved region of 14 nucleotides. Mutational analyses, structural probing, together with footprinting experiments by chemical modification, and Pb2+-induced cleavage showed that this conserved sequence harbours the antibiotic-binding site and forms a stem-loop structure. Moreover, the loop is engaged in a long-range interaction forming a pseudoknot. CONCLUSIONS: A comparison between the novel viomycin-binding motif and the natural RNA target sites for viomycin showed that all these segments form a pseudoknot at the antibiotic-binding site. We therefore conclude that this peptide antibiotic has a strong selectivity for particular RNA pseudoknots.
Asunto(s)
Antibacterianos/metabolismo , Conformación de Ácido Nucleico , ARN/química , ARN/metabolismo , Viomicina/metabolismo , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Enviomicina/análogos & derivados , Enviomicina/metabolismo , Humanos , Plomo/química , Datos de Secuencia Molecular , Mutación , ARN/aislamiento & purificación , Sondas ARNRESUMEN
AcPhe2-tRNA(Phe) which appears in ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. One could readily explain this fact to be the consequence of spontaneous translocation. However, a detailed study of kinetics of puromycin reaction carried out with the use of viomycin (inhibitor of translocation) and the P-site test revealed that, apart from spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction triggers conformational changes in the A-site ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl site of peptidyltransferase center. This is detected functionally as a highly pronounced ability of such a peptidyl-tRNA to react with puromycin.
Asunto(s)
Puromicina/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Sitios de Unión , Cinética , Aminoacil-ARN de Transferencia/química , Viomicina/metabolismoAsunto(s)
Proteínas Bacterianas/metabolismo , Enviomicina/metabolismo , Unión Proteica , Ribosomas/metabolismo , Viomicina/metabolismo , Sistema Libre de Células , Diálisis , Resistencia a Medicamentos , Enviomicina/análogos & derivados , Escherichia coli/metabolismo , Pruebas de Mutagenicidad , Mycobacterium/metabolismo , Proteínas Ribosómicas/metabolismoRESUMEN
The binding of 14C-labelled tuberactinomycin O was analysed in equilibrium dialysis cells. The ionic conditions and the concentration of the labelled drug used in the binding assays allowed the binding of just one drug molecule per non-programmed ribosome. Under these conditions, the occupation of the ribosomal P-site by deacylated tRNAPhe in the presence of poly(U) increased the amount of [14C]tuberactinomycin O bound by a factor of two. Kanamycin, gentamicin and neomycin reduced the binding of tuberactinomycin O, whereas chloramphenicol, tetracycline, streptomycin and puromycin had no effect. A stimulation of the binding of tuberactinomycin O was found upon addition of erythromycin.
Asunto(s)
Enviomicina/metabolismo , Escherichia coli/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Viomicina/metabolismo , Antibacterianos/farmacología , Radioisótopos de Carbono , Enviomicina/análogos & derivados , Escherichia coli/efectos de los fármacos , Ribosomas/efectos de los fármacos , Urea/metabolismoRESUMEN
Binding studies were performed with a [14C]-labelled derivative of viomycin, tuberactinomycin 0 (TUM O). TUM O bound to 30S and 50S subunits. The binding component was the RNA, since ribosomal proteins did not bind the drug. Other RNAs such as tRNA, phage RNA (MS2), and homopolynucleotides also bound the drug. Striking differences in the binding capacity of the various homopolynucleotides were found. Poly(U) bound strongly, poly(G) and poly(C) bound intermediately, whereas poly(A) showed a very low binding. DNA also bound TUM O, although with native DNA the binding was only weak. Finally the effects of viomycin on the assembly in vitro of the 50S subunit from E. coli were tested. A very strong inhibition was found: when the reconstitution was performed at 0.5 x 10(-6) M viomycin the particles formed sedimented at about 50S, but showed a residual activity of less than 10%. The inhibitory power of viomycin with respect to the in vitro assembly is more pronounced than that found in in vitro systems for protein synthesis.