Crystal structure of a complex between the aminoglycoside tobramycin and an oligonucleotide containing the ribosomal decoding a site

Aminoglycoside antibiotics target the decoding aminoacyl site (A site) on the 16S ribosomal RNA and induce miscoding during translation. Here, we present the crystal structure, at 2.54 A resolution, of an RNA oligonucleotide containing the A site sequence complexed to the 4,6-disubstituted 2-deoxystreptamine aminoglycoside tobramycin. The three aminosugar rings making up tobramycin interact with the deep-groove atoms directly or via water molecules and stabilize a fully bulged-out conformation of adenines A(1492) and A(1493). The comparison between this structure and the one previously solved in the presence of paromomycin confirms the importance of the functional groups on the common neamine part of these two antibiotics for binding to RNA. Furthermore, the analysis of the present structure provides a molecular explanation to some of the resistance mechanisms that have spread among bacteria and rendered aminoglycoside antibiotics inefficient.     

Molecular dynamics study of the ribosomal A-site

Many aminoglycosidic antibiotics target the A-site of 16S RNA in the small ribosomal subunit and affect the fidelity of protein translation in bacteria. Upon binding, aminoglycosides displace two adenines (A1492 and A1493 for E. coli numbering) that are involved in tRNA anticodon loop recognition. The major difference in the aminoglycosidic binding site between the prokaryota and eukaryota is an adenine into guanine substitution in the position 1408. This mutation likely affects the dynamics of near A1492 and A1493 and hinders the binding of aminoglycosides to eukaryotic ribosomes. With multiple 20 ns long all-atom molecular dynamics simulations, we study the flexibility of a 22 nucleotide RNA fragment which mimics the aminoglycosidic binding site. Simulations are carried out for both native and A1408G mutated RNA as well as for their complexes with aminoglycosidic representative paromomycin. We observe intra- and extrahelical configurations of A1492 and A1493, which differ between the prokaryotic and the mutated structure. We obtain configurations of the A-site that are also observed in the NMR and crystal structures. Our studies show the differences in the internal mobility of the A-site, as well as that in ion and water density distributions inside of the binding cleft, between the prokaryotic and mutated RNA. We also compare the performance of two force field parameters for RNA, Amber and Charmm.     

Interactions of designer antibiotics and the bacterial ribosomal aminoacyl-tRNA site

The X-ray crystal structures for the complexes of three designer antibiotics, compounds 1, 2, and 3, bound to two models for the ribosomal aminoacyl-tRNA site (A site) at 2.5-3.0 Angstroms resolution and that of neamine at 2.8 Angstroms resolution are described. Furthermore, the complex of antibiotic 1 bound to the A site in the entire 30S ribosomal subunit of Thermus thermophilus is reported at 3.8 Angstroms resolution. Molecular dynamics simulations revealed that the designer compounds provide additional stability to bases A1492 and A1493 in their extrahelical forms. Snapshots from the simulations were used for free energy calculations, which revealed that van der Waals and hydrophobic effects were the driving forces behind the binding of designer antibiotic 3 when compared to the parental neamine.     

Exploring assembly energetics of the 30S ribosomal subunit using an implicit solvent approach

To explore the relationship between the assembly of the 30S ribosomal subunit and interactions among the constituent components, 16S RNA and proteins, relative binding free energies of the T. thermophilus 30S proteins to the 16S RNA were studied based on an implicit solvent model of electrostatic, nonpolar, and entropic contributions. The late binding proteins in our assembly map were found not to bind to the naked 16S RNA. The 5' domain early kinetic class proteins, on average, carry the highest positive charge, get buried the most upon binding to 16S RNA, and show the most favorable binding. Some proteins (S10/S14, S6/S18, S13/S19) have more stabilizing interactions while binding as dimers. Our computed assembly map resembles that of E. coli; however, the central domain path is more similar to that of A. aeolicus, a hyperthermophilic bacteria.     

Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences

Aminoglycoside antibiotics bind specifically to a conserved sequence of the 16S ribosomal RNA (rRNA) A site and interfere with protein synthesis. One model for the mechanism underlying the deleterious effects of aminoglycosides on protein synthesis invokes a drug-induced conformational change in the rRNA that involves the destacking of two adenine residues (A1492 and A1493 in Escherichia coli) at the A site. We describe here a fluorescence-based approach for detecting and characterizing this drug-induced conformational change in the target rRNA. In this approach, we insert the fluorescent base analogue 2-aminopurine in place of A1492 in an E. coli 16S rRNA A-site model oligonucleotide (EcWT) as well as in a mutant form of this oligomer (A1408G) in which A1408 has been replaced with a guanine. The presence of guanine at 1408 instead of adenine represents one of the major sequence differences between prokaryotic and eukaryotic A sites, with the latter A sites being resistant to the deleterious effects of aminoglycosides. Binding of the aminoglycoside paromomycin to the 2AP-substituted forms of EcWT and A1408G induced changes in fluorescence quantum yield consistent with drug-induced base destacking in EcWT but not A1408G. Isothermal titration calorimetry studies reveal that paromomycin binds to the EcWT duplex with a 31-fold higher affinity than the A1408G duplex, with this differential affinity being enthalpic in origin. In the aggregate, these observations are consistent with both rRNA binding affinity and drug-induced base destacking being important determinants in the prokaryotic specificity of aminoglycosides. Combining fluorescence quantum yield and lifetime data allows for quantification of the extent of drug-induced base destacking, thereby providing a convenient tool for evaluating the relative impacts of both novel and existing A-site targeting ligands on rRNA conformation and potentially for predicting relative antibiotic activities and specificities.     

Is tRNA binding or tRNA mimicry mandatory for translation factors?

tRNA is the adaptor in the translation process. The ribosome has three sites for tRNA, the A-, P-, and E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit and the peptidyl transfer site on the large or 50S subunit. The possibility that translation release factors could mimic tRNA has been discussed for a long time, since their function is very similar to that of tRNA. They identify stop codons of the mRNA presented in the decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the peptidyl transfer site. The structures of eubacterial release factors are not yet known, and the first example of tRNA mimicry was discovered when elongation factor G (EF-G) was found to have a closely similar shape to a complex of elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of the tRNA shape is seen in ribosome recycling factor (RRF). The number of proteins mimicking tRNA is rapidly increasing. This primarily concerns translation factors. It is now evident that in some sense they are either tRNA mimics, GTPases or possibly both.     

分子动力学模拟研究A1408G突变对核糖体16S rRNA片段的影响

利用分子动力学模拟方法, 研究了原核生物核糖体小亚基中的16S rRNA片段与氨基糖苷类抗生素巴龙霉素复合物结构的柔性. 结果表明, 16S rRNA片段中的1408位点的腺嘌呤(A)突变为鸟嘌呤(G), 改变了与tRNA中反密码子环识别相关的2个腺嘌呤A1492和A1493的空间构象, 阻碍了氨基糖苷类抗生素与核糖体的结合, 从而影响原核生物蛋白转录过程. 模拟结果与实验测定的晶体结构相吻合, 可为基于核糖体16S rRNA的药物分子设计提供较可靠的结构信息.     

Aminoglycosides modified by resistance enzymes display diminished binding to the bacterial ribosomal aminoacyl-tRNA site

Understanding the basic principles that govern RNA binding by aminoglycosides is important for the design of new generations of antibiotics that do not suffer from the known mechanisms of drug resistance. With this goal in mind, we examined the binding of kanamycin A and four derivatives (the products of enzymic turnovers of kanamycin A by aminoglycoside-modifying enzymes) to a 27 nucleotide RNA representing the bacterial ribosomal A site. Modification of kanamycin A functional groups that have been directly implicated in the maintenance of specific interactions with RNA led to a decrease in affinity for the target RNA. Overall, the products of reactions catalyzed by aminoglycoside resistance enzymes exhibit diminished binding to the A site of bacterial 16S rRNA, which correlates well with a loss of antibacterial ability in resistant organisms that harbor these enzymes.     

Unifying the Aminohexopyranose‐ and Peptidyl‐Nucleoside Antibiotics: Implications for Antibiotic Design

In search of new anti‐tuberculars compatible with anti‐retroviral therapy we re‐identified amicetin as a lead compound. Amicetin's binding to the 70S ribosomal subunit of Thermus thermophilus (Tth) has been unambiguously determined by crystallography and reveals it to occupy the peptidyl transferase center P‐site of the ribosome. The amicetin binding site overlaps significantly with that of the well‐known protein synthesis inhibitor balsticidin S. Amicetin, however, is the first compound structurally characterized to bind to the P‐site with demonstrated selectivity for the inhibition of prokaryotic translation. The natural product‐ribosome structure enabled the synthesis of simplified analogues that retained both potency and selectivity for the inhibition of prokaryotic translation.     

Three-dimensional electron cryomicroscopy of ribosomes

Single particle electron cryomicroscopy is nowadays routinely used to generate three-dimensional structural information of ribosomal complexes without the need of crystallization. A large number of structures of functional important ribosomal complexes have thus been determined using this technique. In E. coli 70S ribosomes all three tRNA binding sites could be localized. The ternary complex of EF-TutRNAGTP that delivers the tRNA to the ribosome was directly visualized in a ribosomal complex blocked by the antibiotic kirromycin. Three different functional states of translocation have been studied and the respective EF-G binding sites have been mapped. The level of resolution achievable with electron cryomicroscopy allows conformational changes in the domain structures of elongation factors to be modelled in terms of rigid body movements. Structural information on eukaryotic ribosomes is also available for yeast and mammalian 80S ribosomes. The structural differences between rabbit 80S and E. coli 70S ribosomes could be interpreted in terms of ribosomal RNA expansion segments in the 18S and 23S RNA. The EF-G homologue EF2 was mapped analysing the structure of an 80SEF2sodarin complex and most recently the binding of a hepatitis C virus IRES element to a yeast 40S subunit has been studied. The first electron cryomicroscopical 3D reconstructions have further been used to overcome the initial phasing problems in X-ray crystallographic studies of the ribosome facilitating structure determination of the recent atomic resolution structures of the 30S and 50S ribosomal subunits. In turn, the knowledge of the atomic structure of the ribosome makes detailed interpretations of cryo-EM maps possible at approximately 20 A resolution.     

Unifying the Aminohexopyranose- and Peptidyl-Nucleoside Antibiotics: Implications for Antibiotic Design

In search of new anti-tuberculars compatible with anti-retroviral therapy we re-identified amicetin as a lead compound. Amicetin's binding to the 70S ribosomal subunit of Thermus thermophilus (Tth) has been unambiguously determined by crystallography and reveals it to occupy the peptidyl transferase center P-site of the ribosome. The amicetin binding site overlaps significantly with that of the well-known protein synthesis inhibitor balsticidin S. Amicetin, however, is the first compound structurally characterized to bind to the P-site with demonstrated selectivity for the inhibition of prokaryotic translation. The natural product-ribosome structure enabled the synthesis of simplified analogues that retained both potency and selectivity for the inhibition of prokaryotic translation.     

Aminoglycoside complexation with a DNA.RNA hybrid duplex: the thermodynamics of recognition and inhibition of RNA processing enzymes

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA > DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.     

A complete mapping of the positions of the proteins in the small ribosomal subunit of escherichia coli

The relative positions of the centers of mass of the 21 proteins of the 30S ribosomal subunit from Escherichia coli have been determined by triangulation using neutron scattering measurements in solution. This mapping is the first complete account of the quaternary structure of the small ribosomal subunit to be obtained by any method.     

Specificity of aminoglycoside antibiotics for the A-site of the decoding region of ribosomal RNA

Background: Aminoglycoside antibiotics bind to the A-site of the decoding region of 16S RNA in the bacterial ribosome, an interaction that is probably responsible for their activity. A detailed study of the specificity of aminoglycoside binding to A-site RNA would improve our understanding of their mechanism of antibiotic activity.Results: We have studied the binding specificity of several aminoglycosides with model RNA sequences derived from the 16S ribosomal A-site using surface plasmon resonance. The 4,5-linked (neomycin) class of aminoglycosides showed specificity for wild-type A-site sequences, but the 4,6-linked class (kanamycins and gentamicins), generally showed poor specificity for the same sequences. Methylation of a cytidine in the target RNA, as found in the Escherichia coli ribosome, had negligible effects on aminoglycoside binding.Conclusions: Although both 4,5- and 4,6-linked aminoglycosides target the same ribosomal site, they appear to bind and effect antibiotic activity in different manners. The aminoglycosides might recognize different RNA conformations or the interaction might involve different RNA tertiary structures that are not equally sampled in our ribosome-free model. These results imply that models of ribosomal RNA must be carefully designed if the data are expected to accurately reflect biological activity.     

High-resolution structures of large ribosomal subunits from mesophilic eubacteria and halophilic archaea at various functional States

Structural analysis of the recently determined high resolution structures of the small and the large ribosomal subunits from three bacterial sources, assisted by the medium resolution structure of a complex of the entire ribosome with three tRNAs, led to a quantum jump in our understanding of the process of the translation of the genetic code into proteins. Results of these studies highlighted dynamic aspects of protein biosynthesis; illuminated the modes of action of several antibiotics; indicated strategies adopted by ribosomes for maximizing their functional activity and revealed a wealth of architectural elements, including long tails of proteins penetrating the particle s cores and stabilizing the intricate folds of the RNA chains. Binding of substrate analogues showed that the decoding and the peptide-bond formation are accomplished mainly by RNA. However, several proteins may be functionally relevant in directing the mRNA and in mediating the proper orientation of the tRNA molecules within the ribosomal rRNA frame. Elements involved in intersubunit contacts or in substrate binding are inherently flexible, but maintain well-ordered characteristic conformations in unbound particles. The ribosomes utilize this conformational variability for optimizing their efficiency and minimizing non-productive interactions, hence disorder of functionally relevant features may be linked to less active conformations or to far from physiological conditions. Clinically relevant antibiotics bind almost exclusively to rRNA. In the small subunit they affect the decoding accuracy or limit conformational mobility and in the large subunit they either interfere with substrate binding, by interacting with components of the peptidyl transferase cavity, or hinder the progression of the growing peptide chain.     

Structural basis for contrasting activities of ribosome binding thiazole antibiotics

Thiostrepton and micrococcin inhibit protein synthesis by binding to the L11 binding domain (L11BD) of 23S ribosomal RNA. The two compounds are structurally related, yet they produce different effects on ribosomal RNA in footprinting experiments and on elongation factor-G (EF-G)-dependent GTP hydrolysis. Using NMR and an assay based on A1067 methylation by thiostrepton-resistance methyltransferase, we show that the related thiazoles, nosiheptide and siomycin, also bind to this region. The effect of all four antibiotics on EF-G-dependent GTP hydrolysis and EF-G-GDP-ribosome complex formation was studied. Our NMR and biochemical data demonstrate that thiostrepton, nosiheptide, and siomycin share a common profile, which differs from that of micrococcin. We have generated a three-dimensional (3D) model for the interaction of thiostrepton with L11BD RNA. The model rationalizes the differences between micrococcin and the thiostrepton-like antibiotics interacting with L11BD.     

Initiation and inhibition of protein biosynthesis studies at high resolution

Analysis of the high resolution structure of the small subunit from Thermus thermophilus shed light on its inherent conformational variability and indicated an interconnected network of features allowing concerted movements during translocation. It also showed that conformational rearrangements may be involved in subunit association and that a latch-like movement guarantees processivity and ensures maximized fidelity. Conformational mobility is associated with the binding and the anti association function of initiation factor 3, and antibiotics interfering with prevent the initiation of the biosynthetic process. Proteins stabilize the structure mainly by their long basic extensions that penetrate into the ribosomal RNA. When pointing into the solution, these extensions may have functional roles in binding of non-ribosomal factors participating in the process of protein biosynthesis. In addition, although the decoding center is formed of RNA, proteins seem to serve ancillary functions such as stabilizing ist required conformation and assisting the directionality of the translocation.     

High-performance-liquid chromatography ofThermus aquaticus 50S and 30S ribosomal proteins

Summary The ribosomal 50S and 30S subunit proteins (r-proteins) ofThermus aquaticus have, for the first time, been characterized by size exclusion chromatography (SEC) and by reversed phase high performance liquid chromatography (RPC). To ensure that the best resolution in the RPC was obtained. the elution conditions, such as gradient time, flow rate, temperature, ionic strength of the eluent and the type of stationary phase were optimized. Correlation between experimentally found retention times and those predicted by DryLab G was better than 0.7% over 30 peaks. Protein fractions from RPC runs were desalted and processed by gel electrophoresis so that the ribosomal proteins could be identified by their position on SDS-polyacrylamide gels. The enhanced speed and quality of separation which has been achieved in this study is expected to bring advantaces in experimental work with ribosomal proteins as well as with other biopolymers. In our case the high resolution technique provides a basis for the preparation of a collection of individual ribosomal protein components for future rRNA-protein interaction studies.     

The 3′-End of tRNA and Its Role in Protein Biosynthesis

In the course of protein biosynthesis, the 3′-ends of aminoacyl-tRNA (aa-tRNA) and peptidyl-tRNA specifically interact with macromolecules of the protein biosynthesis machinery. The 3′-end of tRNA consists of an invariant C-C-A single strand. Interaction of the aminoacyl-tRNA 3′-end with elongation factor Tu (EF-Tu) containing bound GTP is necessary for the formation of the aa-tRNA·EF-Tu·GTP complex and, after the complex binds to the ribosome, for the GTP hydrolysis. This process is followed by the specific binding of the aminoacyl-tRNA 3′-end to the aminoacyl (A) site of the ribosome. In this review, a model is proposed that involves Watson-Crick base pairing of the C? C sequence of the aminoacyl-tRNA 3′-end with a specific G? G sequence of the ribosomal 23S RNA. Similarly, peptidyl-tRNA binds with its 3′-end to the peptidyl (P) site of the ribosome. This binding may also involve Watson-Crick base pairing of the C-C-A sequence with a complementary sequence of 23S RNA. It is proposed that peptide bond formation is catalyzed by a functional site of the 23S RNA located near the 3′-ends of aminoacyl-tRNA and peptidyl-tRNA. A model is suggested in which two loops of the 23S RNA, brought into close proximity via folding, are involved both in binding the 3′-ends of the tRNAs and in catalyzing peptide bond formation. This model presumes a dynamic structure for ribosomal RNA, which is modulated by interaction with elongation factors and ribosomal proteins.     

Separation of ribosomal subunits on Trisacryl GF 2000

The separation of rat liver and E. coli ribosomal subunits was attempted on Trisacryl GF 2000. Contrary to experiments with Sepharose 4B and Bio-Gel A-15 the 60S mammalian subunit did not bind to the resin at 4 degrees C but eluted within the column volume ahead of the 40S subunit. Puromycin, however, used to prepare the subunits, which on the agarose gels had eluted at the total column volume, exhibited anomalous retardation on the Trisacryl resin. Trisacryl therefore behaves as the more non-polar resin, and the binding of 60S subunits to agarose gels is a result of hydrophilic interaction.