RESUMO
Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery.
Assuntos
Antibacterianos/química , Antibacterianos/metabolismo , Macrolídeos/química , Macrolídeos/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Deinococcus/química , Deinococcus/metabolismo , Descoberta de Drogas , Sinergismo Farmacológico , Eritromicina/análogos & derivados , Eritromicina/química , Eritromicina/metabolismo , Modelos Moleculares , Estrutura Molecular , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismoRESUMO
Various antibiotics bind to ribosomes at functionally relevant locations such as the peptidyl-transferase center (PTC) and the exit tunnel for nascent proteins. High-resolution structures of antibiotics bound to ribosomal particles from a eubacterium that is similar to pathogens and an archaeon that shares properties with eukaryotes are deciphering subtle differences in these highly conserved locations that lead to drug selectivity and thereby facilitate clinical usage. These structures also show that members of antibiotic families with structural differences might bind to specific ribosomal pockets in different modes dominated by their chemical properties. Similarly, the chemical properties of drugs might govern variations in the nature of seemingly identical mechanisms of drug resistance. The observed variability in binding modes justifies expectations for structural design of improved antibiotic properties.
Assuntos
Antibacterianos/imunologia , Antibacterianos/farmacologia , Modelos Imunológicos , Proteínas Ribossômicas/química , Proteínas Ribossômicas/imunologia , Ribossomos/química , Ribossomos/imunologia , Antibacterianos/química , Sítios de Ligação , Sistemas de Liberação de Medicamentos/métodos , Farmacorresistência Bacteriana/imunologia , Sinergismo Farmacológico , Modelos Químicos , Modelos Moleculares , Ligação Proteica , Ribossomos/efeitos dos fármacos , Sensibilidade e Especificidade , Relação Estrutura-AtividadeRESUMO
The linkage between internal ribosomal symmetry and transfer RNA (tRNA) positioning confirmed positional catalysis of amino-acid polymerization. Peptide bonds are formed concurrently with tRNA-3' end rotatory motion, in conjunction with the overall messenger RNA (mRNA)/tRNA translocation. Accurate substrate alignment, mandatory for the processivity of protein biosynthesis, is governed by remote interactions. Inherent flexibility of a conserved nucleotide, anchoring the rotatory motion, facilitates chirality discrimination and antibiotics synergism. Potential tRNA interactions explain the universality of the tRNA CCA-end and P-site preference of initial tRNA. The interactions of protein L2 tail with the symmetry-related region periphery explain its conservation and its contributions to nascent chain elongation.
Assuntos
Cristalografia por Raios X/métodos , Ribossomos/química , Ribossomos/ultraestrutura , Aminoácidos/química , Antibacterianos/química , Azitromicina/farmacologia , Catálise , Modelos Moleculares , Peptídeos/química , Isoformas de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Especificidade por Substrato , Virginiamicina/farmacologiaRESUMO
Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system). The breaking of this single phosphodiester bond results in a complete cessation of protein biosynthesis and cell death. The inactive E517Q mutant of the catalytic domain of colicin E3 binds to 30S ribosomal subunits of Thermus thermophilus, as demonstrated by an immunoblotting assay. A model structure of the complex of the ribosomal subunit 30S and colicin E3, obtained via docking, explains the role of the catalytic residues, suggests a catalytic mechanism and provides insight into the specificity of the reaction. Furthermore, the model structure suggests that the inhibitory action of bound immunity is due to charge repulsion of this acidic protein by the negatively charged rRNA backbone
Assuntos
Colicinas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Ribossomos/metabolismo , Proteínas de Bactérias/metabolismo , Colicinas/antagonistas & inibidores , Colicinas/genética , Mutação , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Thermus thermophilus/metabolismoRESUMO
Resistance to antibiotics is a major problem in modern therapeutics. Ribosomes, the cellular organelle catalyzing the translation of the genetic code into proteins, are targets for several clinically relevant antibiotics. The ribosomes from eubacteria are excellent pathogen models. High resolution structures of the large and small ribosomal subunits were used as references that allowed unambiguous localization of almost a dozen antibiotic drugs, most of which are clinically relevant. Analyses of these structures showed a great diversity in the antibiotics' modes of action, such as interference with substrate binding, hindrance of the mobility required for the biosynthetic process and the blockage of tunnel which provides the path of exit for nascent proteins. All antibiotics studied by us were found to bind primarily to ribosomal RNA and, except for one allosteric effect, their binding did not cause major conformational changes. Antibiotics of the small ribosomal subunit may hinder tRNA binding, decoding, translocation, and the initiation of the entire biosynthetic process. The large subunit agents may target the GTPase center, interfere with peptide bond formation, or block the entrance of the nascent protein exit tunnel. The overall structure of the peptidyl transferase center and the modes of action of the antibiotic agents indicate that the ribosome serves as a template for proper positioning of tRNAs, rather than participating actively in the catalytic events associated with the creation of peptide bonds.
Assuntos
Antibacterianos/farmacologia , Ribossomos/efeitos dos fármacos , Cristalografia , Farmacorresistência Bacteriana , Macrolídeos , Biossíntese de Proteínas , Conformação Proteica , Ribossomos/química , Tetraciclina/farmacologiaRESUMO
Nascent proteins emerge out of ribosomes through an exit tunnel, which was assumed to be a firmly built passive path. Recent biochemical results, however, indicate that the tunnel plays an active role in sequence-specific gating of nascent chains and in responding to cellular signals. Consistently, modulation of the tunnel shape, caused by the binding of the semi-synthetic macrolide troleandomycin to the large ribosomal subunit from Deinococcus radiodurans, was revealed crystallographically. The results provide insights into the tunnel dynamics at high resolution. Here we show that, in addition to the typical steric blockage of the ribosomal tunnel by macrolides, troleandomycin induces a conformational rearrangement in a wall constituent, protein L22, flipping the tip of its highly conserved beta-hairpin across the tunnel. On the basis of mutations that alleviate elongation arrest, the tunnel motion could be correlated with sequence discrimination and gating, suggesting that specific arrest motifs within nascent chain sequences may induce a similar gating mechanism.
Assuntos
Ribossomos/fisiologia , Ribossomos/ultraestrutura , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/ultraestrutura , Transdução de SinaisRESUMO
Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis. The large subunit creates the peptide bond and provides the path for emerging proteins. The small has key roles in initiating the process and controlling its fidelity. Crystallographic studies on complexes of the small and the large eubacterial ribosomal subunits with substrate analogs, antibiotics, and inhibitors confirmed that the ribosomal RNA governs most of its activities, and indicated that the main catalytic contribution of the ribosome is the precise positioning and alignment of its substrates, the tRNA molecules. A symmetry-related region of a significant size, containing about two hundred nucleotides, was revealed in all known structures of the large ribosomal subunit, despite the asymmetric nature of the ribosome. The symmetry rotation axis, identified in the middle of the peptide-bond formation site, coincides with the bond connecting the tRNA double-helical features with its single-stranded 3' end, which is the moiety carrying the amino acids. This thus implies sovereign movements of tRNA features and suggests that tRNA translocation involves a rotatory motion within the ribosomal active site. This motion is guided and anchored by ribosomal nucleotides belonging to the active site walls, and results in geometry suitable for peptide-bond formation with no significant rearrangements. The sole geometrical requirement for this proposed mechanism is that the initial P-site tRNA adopts the flipped orientation. The rotatory motion is the major component of unified machinery for peptide-bond formation, translocation, and nascent protein progression, since its spiral nature ensures the entrance of the nascent peptide into the ribosomal exit tunnel. This tunnel, assumed to be a passive path for the growing chains, was found to be involved dynamically in gating and discrimination.
Assuntos
Cristalografia por Raios X/métodos , Ribossomos/química , Antibacterianos/química , Domínio Catalítico , Modelos Moleculares , Conformação Molecular , Conformação de Ácido Nucleico , Peptídeos/química , Peptidil Transferases/química , Ligação Proteica , Conformação Proteica , RNA/química , RNA de Transferência/química , Tetraciclina/químicaRESUMO
Crystal structures of tRNA mimics complexed with the large ribosomal subunit of Deinococcus radiodurans indicate that remote interactions determine the precise orientation of tRNA in the peptidyl-transferase center (PTC). The PTC tolerates various orientations of puromycin derivatives and its flexibility allows the conformational rearrangements required for peptide-bond formation. Sparsomycin binds to A2602 and alters the PTC conformation. H69, the intersubunit-bridge connecting the PTC and decoding site, may also participate in tRNA placement and translocation. A spiral rotation of the 3' end of the A-site tRNA around a 2-fold axis of symmetry identified within the PTC suggests a unified ribosomal machinery for peptide-bond formation, A-to-P-site translocation, and entrance of nascent proteins into the exit tunnel. Similar 2-fold related regions, detected in all known structures of large ribosomal subunits, indicate the universality of this mechanism.
Assuntos
Deinococcus/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Conformação Proteica , Aminoacil-RNA de Transferência/química , Proteínas Ribossômicas/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Inibidores da Síntese de Proteínas/química , Inibidores da Síntese de Proteínas/metabolismo , Puromicina/química , Puromicina/metabolismo , Aminoacil-RNA de Transferência/genética , Aminoacil-RNA de Transferência/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Esparsomicina/química , Esparsomicina/metabolismoRESUMO
High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation.