RESUMEN
Multidrug resistance is a global threat as the clinically available potent antibiotic drugs are becoming exceedingly scarce. For example, increasing drug resistance among gram-positive bacteria is responsible for approximately one-third of nosocomial infections. As ribosomes are a major target for these drugs, they may serve as suitable objects for novel development of next-generation antibiotics. Three-dimensional structures of ribosomal particles from Staphylococcus aureus obtained by X-ray crystallography have shed light on fine details of drug binding sites and have revealed unique structural motifs specific for this pathogenic strain, which may be used for the design of novel degradable pathogen-specific, and hence, environmentally friendly drugs.
Asunto(s)
Antibacterianos/síntesis química , Proteínas Bacterianas/química , Diseño de Fármacos , Ribosomas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/metabolismo , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/microbiología , Cristalografía por Rayos X , Deinococcus/efectos de los fármacos , Deinococcus/genética , Deinococcus/metabolismo , Farmacorresistencia Bacteriana Múltiple , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Modelos Moleculares , Ribosomas/metabolismo , Ribosomas/ultraestructura , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Thermus thermophilus/efectos de los fármacos , Thermus thermophilus/genética , Thermus thermophilus/metabolismoRESUMEN
Although the mode of action of the ribosomes, the multi-component universal effective protein-synthesis organelles, has been thoroughly explored, their mere appearance remained elusive. Our earlier comparative structural studies suggested that a universal internal small RNA pocket-like segment called by us the protoribosome, which is still embedded in the contemporary ribosome, is a vestige of the primordial ribosome. Herein, after constructing such pockets, we show using the "fragment reaction" and its analyses by MALDI-TOF and LC-MS mass spectrometry techniques, that several protoribosome constructs are indeed capable of mediating peptide-bond formation. These findings present strong evidence supporting our hypothesis on origin of life and on ribosome's construction, thus suggesting that the protoribosome may be the missing link between the RNA dominated world and the contemporary nucleic acids/proteins life.
Asunto(s)
Origen de la Vida , Proteínas/metabolismo , ARN , Ribosomas , Péptidos/metabolismo , Biosíntesis de Proteínas , ARN/metabolismo , Ribosomas/metabolismoRESUMEN
Giardiasis is a disease caused by the protist Giardia lamblia. As no human vaccines have been approved so far against it, and resistance to current drugs is spreading, new strategies for combating giardiasis need to be developed. The G. lamblia ribosome may provide a promising therapeutic target due to its distinct sequence differences from ribosomes of most eukaryotes and prokaryotes. Here, we report the cryo-electron microscopy structure of the G. lamblia (WB strain) ribosome determined at 2.75 Å resolution. The ribosomal RNA is the shortest known among eukaryotes, and lacks nearly all the eukaryote-specific ribosomal RNA expansion segments. In contrast, the ribosomal proteins are typically eukaryotic with some species-specific insertions/extensions. Most typical inter-subunit bridges are maintained except for one missing contact site. Unique structural features are located mainly at the ribosome's periphery. These may be exploited as target sites for the design of new compounds that inhibit selectively the parasite's ribosomal activity.
Asunto(s)
Giardia lamblia , Giardiasis , Parásitos , Animales , Microscopía por Crioelectrón , Eucariontes/genética , Giardia lamblia/genética , Giardiasis/metabolismo , Humanos , Parásitos/genética , ARN Ribosómico/metabolismo , Ribosomas/metabolismoRESUMEN
Antibiotic resistance is a major threat to global health; this problem can be addressed by the development of new antibacterial agents to keep pace with the evolutionary adaptation of pathogens. Computational approaches are essential tools to this end since their application enables fast and early strategical decisions in the drug development process. We present a rational design approach, in which acylide antibiotics were screened based on computational predictions of solubility, membrane permeability, and binding affinity toward the ribosome. To assess our design strategy, we tested all candidates for in vitro inhibitory activity and then evaluated them in vivo with several antibiotic-resistant strains to determine minimal inhibitory concentrations. The predicted best candidate is synthetically more accessible, exhibits higher solubility and binding affinity to the ribosome, and is up to 56 times more active against resistant pathogens than telithromycin. Notably, the best compounds designed by us show activity, especially when combined with the membrane-weakening drug colistin, against Acinetobacter baumanii, Pseudomonas aeruginosa, and Escherichia coli, which are the three most critical targets from the priority list of pathogens of the World Health Organization.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Macrólidos/farmacología , Colistina/farmacología , Pruebas de Sensibilidad Microbiana/métodosRESUMEN
Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.
Asunto(s)
Macrólidos/química , Micromonospora/química , Antibacterianos/química , Antibacterianos/farmacología , Antiinfecciosos/química , Antiinfecciosos/farmacología , Eritromicina/química , Humanos , Macrólidos/farmacología , Pruebas de Sensibilidad Microbiana , Inhibidores de la Síntesis de la Proteína/farmacología , Ribosomas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/patogenicidadRESUMEN
Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.
Asunto(s)
Euglena gracilis/química , ARN Ribosómico/química , Ribosomas/química , Microscopía por Crioelectrón , Citoplasma/química , Euglena gracilis/genética , Euglena gracilis/metabolismo , Modelos Moleculares , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/químicaRESUMEN
Resistance to antibiotics has become a major threat to modern medicine. The ribosome plays a fundamental role in cell vitality by the translation of the genetic code into proteins; hence, it is a major target for clinically useful antibiotics. We report here the cryo-electron microscopy structures of the ribosome of a pathogenic aminoglycoside (AG)-resistant Pseudomonas aeruginosa strain, as well as of a nonresistance strain isolated from a cystic fibrosis patient. The structural studies disclosed defective ribosome complex formation due to a conformational change of rRNA helix H69, an essential intersubunit bridge, and a secondary binding site of the AGs. In addition, a stable conformation of nucleotides A1486 and A1487, pointing into helix h44, is created compared to a non-AG-bound ribosome. We suggest that altering the conformations of ribosomal protein uL6 and rRNA helix H69, which interact with initiation-factor IF2, interferes with proper protein synthesis initiation.
Asunto(s)
Fibrosis Quística/microbiología , Pseudomonas aeruginosa/ultraestructura , Ribosomas/química , Secuencias de Aminoácidos , Aminoglicósidos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Microscopía por Crioelectrón , Farmacorresistencia Bacteriana , Humanos , Mutación , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/aislamiento & purificación , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Ribosomas/ultraestructuraRESUMEN
Antimicrobial resistance within a wide range of pathogenic bacteria is an increasingly serious threat to global public health. Among these pathogenic bacteria are the highly resistant, versatile and possibly aggressive bacteria, Staphylococcus aureus. Lincosamide antibiotics were proved to be effective against this pathogen. This small, albeit important group of antibiotics is mostly active against Gram-positive bacteria, but also used against selected Gram-negative anaerobes and protozoa. S. aureus resistance to lincosamides can be acquired by modifications and/or mutations in the rRNA and rProteins. Here, we present the crystal structures of the large ribosomal subunit of S. aureus in complex with the lincosamides lincomycin and RB02, a novel semisynthetic derivative and discuss the biochemical aspects of the in vitro potency of various lincosamides. These results allow better understanding of the drugs selectivity as well as the importance of the various chemical moieties of the drug for binding and inhibition.
Asunto(s)
Lincosamidas/farmacología , Subunidades Ribosómicas Grandes Bacterianas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Benzamidas/química , Benzamidas/farmacología , Sitios de Unión , Clindamicina/química , Clindamicina/farmacología , Cristalización , Cristalografía por Rayos X , Farmacorresistencia Microbiana , Galactósidos/química , Galactósidos/farmacología , Enlace de Hidrógeno , Lincomicina/química , Lincomicina/farmacología , Lincosamidas/química , Estructura Molecular , Subunidades Ribosómicas Grandes Bacterianas/ultraestructura , Staphylococcus aureus/ultraestructura , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Two structurally unique ribosomal antibiotics belonging to the orthosomycin family, avilamycin and evernimicin, possess activity against Enterococci, Staphylococci, and Streptococci, and other Gram-positive bacteria. Here, we describe the high-resolution crystal structures of the eubacterial large ribosomal subunit in complex with them. Their extended binding sites span the A-tRNA entrance corridor, thus inhibiting protein biosynthesis by blocking the binding site of the A-tRNA elbow, a mechanism not shared with other known antibiotics. Along with using the ribosomal components that bind and discriminate the A-tRNA-namely, ribosomal RNA (rRNA) helices H89, H91, and ribosomal proteins (rProtein) uL16-these structures revealed novel interactions with domain 2 of the CTC protein, a feature typical to various Gram-positive bacteria. Furthermore, analysis of these structures explained how single nucleotide mutations and methylations in helices H89 and H91 confer resistance to orthosomycins and revealed the sequence variations in 23S rRNA nucleotides alongside the difference in the lengths of the eukaryotic and prokaryotic α1 helix of protein uL16 that play a key role in the selectivity of those drugs. The accurate interpretation of the crystal structures that could be performed beyond that recently reported in cryo-EM models provide structural insights that may be useful for the design of novel pathogen-specific antibiotics, and for improving the potency of orthosomycins. Because both drugs are extensively metabolized in vivo, their environmental toxicity is very low, thus placing them at the frontline of drugs with reduced ecological hazards.
Asunto(s)
Aminoglicósidos/farmacología , Proteínas Bacterianas/efectos de los fármacos , Sitios de Unión/efectos de los fármacos , Oligosacáridos/farmacología , ARN de Transferencia/efectos de los fármacos , Proteínas Ribosómicas/efectos de los fármacos , Aminoglicósidos/química , Antibacterianos/farmacología , Cristalografía por Rayos X , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Bacterias Grampositivas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Oligosacáridos/química , Biosíntesis de Proteínas/efectos de los fármacos , ARN Ribosómico , ARN Ribosómico 23S/efectos de los fármacos , ARN Ribosómico 23S/genética , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
The emergence of bacterial multidrug resistance to antibiotics threatens to cause regression to the preantibiotic era. Here we present the crystal structure of the large ribosomal subunit from Staphylococcus aureus, a versatile Gram-positive aggressive pathogen, and its complexes with the known antibiotics linezolid and telithromycin, as well as with a new, highly potent pleuromutilin derivative, BC-3205. These crystal structures shed light on specific structural motifs of the S. aureus ribosome and the binding modes of the aforementioned antibiotics. Moreover, by analyzing the ribosome structure and comparing it with those of nonpathogenic bacterial models, we identified some unique internal and peripheral structural motifs that may be potential candidates for improving known antibiotics and for use in the design of selective antibiotic drugs against S. aureus.
Asunto(s)
Ribosomas/metabolismo , Staphylococcus aureus/metabolismo , Conformación Proteica , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismoRESUMEN
Experimental evidence suggests the existence of an RNA molecular prebiotic entity, called by us the "protoribosome," which may have evolved in the RNA world before evolution of the genetic code and proteins. This vestige of the RNA world, which possesses all of the capabilities required for peptide bond formation, seems to be still functioning in the heart of all of the contemporary ribosome. Within the modern ribosome this remnant includes the peptidyl transferase center. Its highly conserved nucleotide sequence is suggestive of its robustness under diverse environmental conditions, and hence on its prebiotic origin. Its twofold pseudosymmetry suggests that this entity could have been a dimer of self-folding RNA units that formed a pocket within which two activated amino acids might be accommodated, similar to the binding mode of modern tRNA molecules that carry amino acids or peptidyl moieties. Using quantum mechanics and crystal coordinates, this work studies the question of whether the putative protoribosome has properties necessary to function as an evolutionary precursor to the modern ribosome. The quantum model used in the calculations is density functional theory--B3LYP/3-21G*, implemented using the kernel energy method to make the computations practical and efficient. It occurs that the necessary conditions that would characterize a practicable protoribosome--namely (i) energetic structural stability and (ii) energetically stable attachment to substrates--are both well satisfied.
Asunto(s)
Evolución Biológica , ARN/química , ARN/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Modelos Moleculares , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , Teoría Cuántica , Pliegue del ARN , TermodinámicaRESUMEN
We have developed a collagen-mRNA platform for controllable protein production that is intended to be less prone to the problems associated with commonly used mRNA therapy as well as with collagen skin-healing procedures. A collagen mimic was constructed according to a recombinant method and was used as scaffold for translating mRNA chains into proteins. Cysteines were genetically inserted into the collagen chain at positions allowing efficient ribosome translation activity while minimizing mRNA misfolding and degradation. Enhanced green fluorescence protein (eGFP) mRNA bound to collagen was successfully translated by cell-free Escherichia coli ribosomes. This system enabled an accurate control of specific protein synthesis by monitoring expression time and level. Luciferase-mRNA was also translated on collagen scaffold by eukaryotic cell extracts. Thus we have demonstrated the feasibility of controllable protein synthesis on collagen scaffolds by ribosomal machinery.
Asunto(s)
Sistema Libre de Células , Colágeno/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Sistema Libre de Células/metabolismo , Colágeno/química , Escherichia coli/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Luciferasas/análisis , Luciferasas/genética , Sustancias Luminiscentes/análisis , Sustancias Luminiscentes/metabolismo , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/genética , Multimerización de Proteína , Estabilidad Proteica , ARN Mensajero/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The structures of the large ribosomal subunit of Deinococcus radiodurans (D50S) in complex with the antibiotic lankamycin (3.2 Å) and a double antibiotic complex of lankamycin and lankacidin C (3.45 Å) have been determined, in continuation of previous crystallographic studies on lankacidin-D50S complex. These two drugs have been previously reported to inhibit ribosomal function with mild synergistic effect. Lankamycin, a member of the macrolide family, binds in a similar manner to erythromycin. However, when in complex with lankacidin, lankamycin is located so that it can form interactions with lankacidin in the adjacent ribosomal binding site. When compared to the well-documented synergistic antibiotics, Streptogramins A and B, the pair of lankacidin and lankamycin bind in similar sites, the peptidyl transferase center and nascent peptide exit tunnel, respectively. Herein, we discuss the structural basis for antibiotic synergism and highlight the key factors involved in ribosomal inhibition.
Asunto(s)
Antibacterianos/química , Eritromicina/análogos & derivados , Macrólidos/química , Modelos Moleculares , Subunidades Ribosómicas Grandes/química , Sitios de Unión/genética , Cristalografía , Huella de ADN , Sinergismo Farmacológico , Eritromicina/química , Concentración 50 Inhibidora , Estructura Molecular , ARN Ribosómico 23S/genética , Difracción de Rayos XRESUMEN
Leishmania is the causative agent of cutaneous and visceral diseases affecting millions of individuals worldwide. Pseudouridine (Ψ), the most abundant modification on rRNA, changes during the parasite life cycle. Alterations in the level of a specific Ψ in helix 69 (H69) affected ribosome function. To decipher the molecular mechanism of this phenotype, we determine the structure of ribosomes lacking the single Ψ and its parental strain at â¼2.4-3 Å resolution using cryo-EM. Our findings demonstrate the significance of a single Ψ on H69 to its structure and the importance for its interactions with helix 44 and specific tRNAs. Our study suggests that rRNA modification affects translation of mRNAs carrying codon bias due to selective accommodation of tRNAs by the ribosome. Based on the high-resolution structures, we propose a mechanism explaining how the ribosome selects specific tRNAs.
Asunto(s)
Seudouridina , ARN de Transferencia , Ribosomas , Seudouridina/metabolismo , Ribosomas/metabolismo , ARN de Transferencia/metabolismo , ARN de Transferencia/genética , Leishmania/metabolismo , Leishmania/genética , Microscopía por Crioelectrón , ARN Ribosómico/metabolismo , ARN Ribosómico/química , ARN Ribosómico/genética , Conformación de Ácido Nucleico , Modelos MolecularesRESUMEN
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.
Asunto(s)
Antibacterianos/química , Antibacterianos/metabolismo , Macrólidos/química , Macrólidos/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Deinococcus/química , Deinococcus/metabolismo , Descubrimiento de Drogas , Sinergismo Farmacológico , Eritromicina/análogos & derivados , Eritromicina/química , Eritromicina/metabolismo , Modelos Moleculares , Estructura Molecular , Subunidades Ribosómicas Grandes Bacterianas/química , Subunidades Ribosómicas Grandes Bacterianas/metabolismoRESUMEN
Assessment of structure-activity relationships for anti-protozoan activity revealed a strategy for preparing potent anisomycin derivatives with reduced host toxicity. Thirteen anisomycin analogs were synthesized by modifying the alcohol, amine, and aromatic functional groups. Examination of anti-protozoal activity against various strains of Leishmania and cytotoxicity against leucocytes with comparison against the parent natural product demonstrated typical losses of activity with modifications of the alcohol, amine, and aromatic meta-positions. On the other hand, the para-phenol moiety of anisomycin proved an effective location for introducing substituents without significant loss of anti-protozoan potency. An entry point for differentiating activity against Leishmania versus host has been uncovered by this systematic study.
RESUMEN
Trypanosomes are protozoan parasites that cycle between insect and mammalian hosts and are the causative agent of sleeping sickness. Here, we describe the changes of pseudouridine (Ψ) modification on rRNA in the two life stages of the parasite using four different genome-wide approaches. CRISPR-Cas9 knock-outs of all four snoRNAs guiding Ψ on helix 69 (H69) of the large rRNA subunit were lethal. A single knock-out of a snoRNA guiding Ψ530 on H69 altered the composition of the 80S monosome. These changes specifically affected the translation of only a subset of proteins. This study correlates a single site Ψ modification with changes in ribosomal protein stoichiometry, supported by a high-resolution cryo-EM structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.
Asunto(s)
Parásitos , Trypanosoma brucei brucei , Animales , Parásitos/genética , Trypanosoma brucei brucei/metabolismo , Seudouridina/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Ribosomas/metabolismo , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Mamíferos/genéticaRESUMEN
Ribosomes, the cellular organelles translating the genetic code to proteins, are assemblies of RNA chains and many proteins (RPs) arranged in precise fine-tuned interwoven structures. Mutated ribosomal genes cause ribosomopathies, including Diamond Blackfan anemia (DBA, a rare heterogeneous red-cell aplasia connected to ribosome malfunction) or failed biogenesis. Combined bioinformatical, structural, and predictive analyses of potential consequences of possibly expressed mutations in eS19, the protein product of the highly mutated RPS19, suggest that mutations in its exposed surface could alter its positioning during assembly and consequently prevent biogenesis, implying a natural selective strategy to avoid malfunctions in ribosome assembly. A search for RPS19 pseudogenes indicated > 90% sequence identity with the wild-type, hinting at its expression in cases of absent or truncated gene products.
Asunto(s)
Anemia de Diamond-Blackfan , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/metabolismo , Humanos , Mutación/genética , ARN/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
The ribosome, a multicomponent assembly consisting of RNA and proteins, is a pivotal macromolecular machine that translates the genetic code into proteins. The large ribosomal subunit rRNA helix 68 (H68) is a key element in the protein synthesis process, as it coordinates the coupled movements of the actors involved in translocation, including the tRNAs and L1 stalk. Examination of cryo-electron microscopy (cryo-EM) structures of ribosomes incubated for various time durations at physiological temperatures led to the identification of functionally relevant H68 movements. These movements assist the transition of the L1 stalk between its open and closed states. H68 spatial flexibility and its significance to the protein synthesis process were confirmed through its effective targeting with antisense PNA oligomers. Our results suggest that H68 is actively involved in ribosome movements that are central to the elongation process. IMPORTANCE The mechanism that regulates the translocation step in ribosomes during protein synthesis is not fully understood. In this work, cryo-EM techniques used to image ribosomes from Staphylococcus aureus after incubation at physiological temperature allowed the identification of a conformation of the helix 68 that has never been observed so far. We then propose a mechanism in which such helix, switching between two different conformations, actively coordinates the translocation step, shedding light on the dynamics of ribosomal components. In addition, the relevance of helix 68 to ribosome function and its potential as an antibiotic target was proved by inhibiting Staphylococcus aureus ribosomes activity in vitro using oligomers with sequence complementarity.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas , Microscopía por Crioelectrón/métodos , Modelos Moleculares , ARN de Transferencia/metabolismo , Ribosomas/metabolismoRESUMEN
Clinically relevant antibiotics that target the ribosomal peptidyl transferase center (PTC), a highly conserved ribosomal region, exert their inhibitory action by exploiting the flexibility of PTC nucleotides, which trigger modulations of the shape of the antibiotic binding pocket. Resistance to these antibiotics was observed clinically and in vitro. Based on the crystal structures of the large ribosomal subunit from eubacterium suitable to represent pathogens in complex with these antibiotics, it was found that all nucleotides mediating resistance to PTC antibiotics cluster on one side of the PTC. Over half of the nucleotides affecting resistance reside in regions of lower sequence conservation, and are too distal to make Van der Waals interactions with the bound drugs. Alterations of the identity of these nucleotides may not lethally affect ribosome function, but can hamper antibiotic binding through changes in the conformation and flexibility of specific PTC nucleotides. Comparative analysis revealed properties likely to lead to cross-resistance and enabled their parameterization. As the same nucleotides are frequently involved in resistance to more than a single family of antibiotics, the common pattern explains medically observed cross-resistance to PTC antibiotics and suggests the potential for a wider clinical threat.