Aminoglycoside Binding Research Articles

Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA

Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome’s subunits by binding to Helix 69 (H69) of 23S rRNA. The differential binding of various aminoglycosides to the chemically synthesized terminal domains of the Escherichia coli and human H69 has been characterized using spectroscopy, calorimetry and NMR. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin B and tobramycin than for paromomycin (Kds = 0.3 ± 0.1, 0.2 ± 0.2 and 5.4 ± 1.1 µM, respectively). The binding of streptomycin was too weak to assess. In contrast to the E. coli H69, the human 28S rRNA H69 had a considerable decrease in affinity for the antibiotics, an important validation of the bacterial target. The three conserved pseudouridine modifications (Ψ1911, Ψ1915, Ψ1917) occurring in the loop of the E. coli H69 affected the dissociation constant, but not the stoichiometry for the binding of paromomycin (Kd = 2.6 ± 0.1 µM). G1906 and G1921, observed by NMR spectrometry, figured predominantly in the aminoglycoside binding to H69. The higher affinity of the E. coli H69 for neomycin B and tobramycin, as compared to paromomycin and streptomycin, indicates differences in the efficacy of the aminoglycosides.

Triple Recognition of B-DNA by a Neomycin−Hoechst 33258−Pyrene Conjugate

Recent developments have indicated that aminoglycoside binding is not limited to RNA, but to nucleic acids that, like RNA, adopt conformations similar to its A-form. We further sought to expand the utility of aminoglycoside binding to B-DNA structures by conjugating neomycin, an aminoglycoside antibiotic, with the B-DNA minor groove binding ligand Hoechst 33258. Envisioning a dual groove binding mode, we have extended the potential recognition process to include a third, intercalative moiety. Similar conjugates, which vary in the number of binding moieties but maintain identical linkages to allow direct comparisons to be made, have also been prepared. We report herein novel neomycin- and Hoechst 33258-based conjugates developed in our laboratories for exploring the recognition potential with B-DNA. Spectroscopic studies such as UV melting, differential scanning calorimetry, isothermal fluorescence titrations, and circular dichroism together illustrate the triple recognition of the novel conjugate containing neomycin, Hoechst 33258, and pyrene. This study represents the first example of DNA molecular recognition capable of minor versus major groove recognition in conjunction with intercalation.

Elucidating Protein Binding Mechanisms by Variable‐c ITC

Elucidating Protein Binding Mechanisms by Variable‐<i>c</i> ITC

Aminoglycoside activity observed on single pre-translocation ribosome complexes

Aminoglycoside-class antibiotics bind directly to ribosomal RNA, imparting pleiotropic effects on ribosome function. Despite in-depth structural investigations of aminoglycoside-RNA oligonucleotide and aminoglycoside-ribosome interactions, mechanisms explaining the unique ribosome inhibition profiles of chemically similar aminoglycosides remain elusive. Here, using single-molecule fluorescence resonance energy transfer (smFRET) methods, we show that high-affinity aminoglycoside binding to the conserved decoding site region of the functional pre-translocation ribosome complex specifically remodels the nature of intrinsic dynamic processes within the particle. The extents of these effects, which are distinct for each member of the aminoglycoside class, strongly correlate with their inhibition of EF-G-catalyzed translocation. Neomycin, a 4,5-linked aminoglycoside, binds with lower affinity to one or more secondary binding sites, mediating distinct structural and dynamic perturbations that further enhance translocation inhibition. These new insights help explain why closely related aminoglycosides elicit pleiotropic translation activities and demonstrate the potential utility of smFRET as a tool for dissecting the mechanisms of antibiotic action.

NMR Detected Hydrogen−Deuterium Exchange Reveals Differential Dynamics of Antibiotic- and Nucleotide-Bound Aminoglycoside Phosphotransferase 3′-IIIa

In this work, hydrogen-deuterium exchange detected by NMR spectroscopy is used to determine the dynamic properties of the aminoglycoside phosphotransferase 3'-IIIa (APH), a protein of intense interest due to its involvement in conferring antibiotic resistance to both gram negative and gram positive microorganisms. This represents the first characterization of dynamic properties of an aminoglycoside-modifying enzyme. Herein we describe in vitro dynamics of apo, binary, and ternary complexes of APH with kanamycin A, neomycin B, and metal-nucleotide. Regions of APH in different complexes that are superimposable in crystal structures show remarkably different dynamic behavior. A complete exchange of backbone amides is observed within the first 15 h of exposure to D(2)O in the apo form of this 31 kDa protein. Binding of aminoglycosides to the enzyme induces significant protection against exchange, and approximately 30% of the amides remain unexchanged up to 95 h after exposure to D(2)O. Our data also indicate that neomycin creates greater solvent protection and overall enhanced structural stability to APH than kanamycin. Surprisingly, nucleotide binding to the enzyme-aminoglycoside complex increases solvent accessibility of a number of amides and is responsible for destabilization of a nearby beta-sheet, thus providing a rational explanation for previously observed global thermodynamic parameters. Our data also provide a molecular basis for broad substrate selectivity of APH.

Hydrogen‐Deuterium Exchange Reveals Antibiotic Resistance Enzyme Aminoglycoside Phosphotransferase 3′‐IIIa as Intrinsically Unstructured Protein

Hydrogen deuterium (H/D) exchange detected by NMR spectroscopy is used to determine the dynamic properties of the aminoglycoside phosphotransferase 3′‐IIIa (APH), which confer resistance to aminoglycoside antibiotics. This work represents the first characterization of dynamic properties of an aminoglycoside modifying enzyme. H/D exchange experiments performed with apo, binary and ternary complexes of APH with kanamycin A, neomycin B, and metal‐nucleotide showed that regions of APH in different complexes that are superimposable in crystal structures show remarkably different dynamic behavior. A complete exchange of backbone amides is observed within the first 15 hours of exposure to D2O in the apo form of this 31 kDa protein. Binding of aminoglycosides to the enzyme induces significant protection against exchange and ~35% of the amides remain unexchanged up to 90 hours after exposure to D2O. Our data also indicate that neomycin creates greater solvent protection and overall enhanced structural stability to APH than kanamycin. Surprisingly, nucleotide binding to enzyme‐aminoglycoside complex increases solvent accessibility of a number of amides and is responsible for destabilization of a nearby β‐sheet thus providing a rational explanation for previously observed global thermodynamic parameters. Our data also provide molecular basis for broad substrate selectivity of APH.

Binding of aminoglycoside antibiotics to helix 69 of 23S rRNA

Aminoglycosides antibiotics negate dissociation and recycling of the bacterial ribosome's subunits by binding to Helix 69 (H69) of 23S rRNA. Aminoglycoside binding to the chemically synthesized terminal domains of the E. coli and human H69 was detected and quantified using UV‐monitored thermal denaturation, circular dichroism spectrapolarimetry, and isothermal titration calorimetry. The unmodified E. coli H69 hairpin exhibited a significantly higher affinity for neomycin and tobramycin than for paromomycin, while the binding of streptomycin was too weak to assess. The three conserved pseudouridine modifications (Ψ1911, Ψ1915, Ψ1917) occurring in the loop of the E. coli H69 did not significantly effect the dissociation constant or stoichiometry for the binding of paromomycin. In contrast, the human 28S rRNA H69 had a considerably decreased affinity for the antibiotics. The higher affinity of the bacterial 23S rRNA H69 for aminoglycosides is an important validation of the antibiotic target. The higher affinity of the E. coli H69 for neomycin and tobramycin as compared to paromomycin and streptomycin indicates differences in the efficacy of the aminoglycosides as anti‐microbial agents.

Aminoglycoside antibiotics: A-site specific binding to 16S

The A-site of 16S rRNA, which is a part of the 30S ribosomal subunit involved in prokaryotic translation, is a well known aminoglycoside binding site. Full characterization of the conformational changes undergone at the A-site upon aminoglycoside binding is essential for development of future RNA/drug complexes; however, the massiveness of 16S makes this very difficult. Recently, studies have found that a 27 base RNA construct (16S 27) that comprises the A-site subdomain of 16S behaves similarly to the whole A-site domain. ESI-MS, ion mobility and molecular dynamics methods were utilized in this study to analyze the A-site of 16S 27 before and after the addition of ribostamycin (R), paromomycin (P) and lividomycin (L). The ESI mass spectrum for 16S 27 alone illustrated both single-stranded 16S 27 and double-stranded (16S 27) 2 complexes. Upon aminoglycoside addition, the mass spectra showed that only one aminoglycoside binds to 16S 27, while either one or two bind to (16S 27) 2. Ion mobility measurements and molecular dynamics calculations were utilized in determining the solvent-free structures of the 16S 27 and (16S 27) 2 complexes. These studies found 16S 27 in a hairpin conformation while (16S 27) 2 existed as a cruciform. Only one aminoglycoside binds to the single A-site of the 16S 27 hairpin and this attachment compresses the hairpin. Since two A-sites exist for the (16S 27) 2 cruciform, either one or two aminoglycosides may bind. The aminoglycosides compress the A-sites causing the cruciform with just one aminoglycoside bound to be larger than the cruciform with two bound. Non-specific binding was not observed in any of the aminoglycoside/16S 27 complexes.

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.

Guanidinoneomycin B recognition of an HIV-1 RNA helix.

Aminoglycoside antibiotics are small-molecule drugs that bind RNA. The affinity and specificity of aminoglycoside binding to RNA can be increased through chemical modification, such as guanidinylation. Here, we report the binding of guanidinoneomycin B (GNB) to an RNA helix from the HIV-1 frameshift site. The binding of GNB increases the melting temperature (T(m)) of the frameshift-site RNA by at least 10 degrees C, to a point at which a melting transition is not even observed in 2 M urea. A structure of the complex was obtained by using multidimensional heteronuclear NMR spectroscopic methods. We also used a novel paramagnetic-probe assay to identify the site of GNB binding to the surface of the RNA. GNB makes major-groove contacts to two sets of Watson-Crick bases and is in van der Waals contact with a highly structured ACAA tetraloop. Rings I and II of GNB fit into the major groove and form the binding interface with the RNA, whereas rings III and IV are exposed to the solvent and disordered. The binding of GNB causes a broadening of the major groove across the binding site.

Detection of Specific Solvent Rearrangement Regions of an Enzyme: NMR and ITC Studies with Aminoglycoside Phosphotransferase(3‘)-IIIa

This work describes differential effects of solvent in complexes of the aminoglycoside phosphotransferase(3')-IIIa (APH) with different aminoglycosides and the detection of change in solvent structure at specific sites away from substrates. Binding of kanamycins to APH occurs with a larger negative DeltaH in H2O relative to D2O (DeltaDeltaH(H2O-D2O) < 0), while the reverse is true for neomycins. Unusually large negative DeltaCp values were observed for binding of aminoglycosides to APH. DeltaCp for the APH-neomycin complex was -1.6 kcal x mol(-1) x deg(-1). A break at 30 degrees C was observed in the APH-kanamycin complex yielding DeltaCp values of -0.7 kcal x mol(-1) x deg(-1) and -3.8 kcal x mol(-1) x deg(-1) below and above 30 degrees C, respectively. Neither the change in accessible surface area (DeltaASA) nor contributions from heats of ionization were sufficient to explain the large negative DeltaCp values. Most significantly, 15N-1H HSQC experiments showed that temperature-dependent shifts of the backbone amide protons of Leu 88, Ser 91, Cys 98, and Leu143 revealed a break at 30 degrees C only in the APH-kanamycin complex in spectra collected between 21 degrees C and 38 degrees C. These amino acids represent solvent reorganization sites that experience a change in solvent structure in their immediate environment as structurally different ligands bind to the enzyme. These residues were away from the substrate binding site and distributed in three hydrophobic patches in APH. Overall, our results show that a large number of factors affect DeltaCp and binding of structurally different ligand groups cause different solvent structure in the active site as well as differentially affecting specific sites away from the ligand binding site.

Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion

Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop–loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug–RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (Kd ∼ 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (Kd ∼ 1.6 µM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop–loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

Streptomycin and gentamicin have no immediate effect on outer hair cell electromotility

The cochlear outer hair cell (OHC), which plays a crucial role in mammalian hearing through its unique voltage-dependent motility, has been established as a primary target of the ototoxicity of aminoglycoside antibiotics. These polycationic drugs are also known to block a wide variety of ion channels, purinergic ionotropic channels, and nicotinic ACh receptors in hair cells in vitro. The OHC motor protein, prestin, is a voltage-sensitive transmembrane protein containing several negatively charged residues on both intra- and extracellular surface. The acidic sites may be susceptible to polycationic-charged aminoglycoside binding, which may result in disruption of motility. We attempted to examine whether aminoglycosides such as streptomycin and gentamicin could affect OHC motility and its electrical signature, the nonlinear capacitance (NLC) in adult gerbil OHCs. Somatic motility and NLC were measured under the whole-cell voltage-clamp mode. Streptomycin and gentamicin were applied extracellularly or intracellularly. Results show that streptomycin and gentamicin did not change either the magnitude of motility or the NLC. Theses results suggest that, although streptomycin and gentamicin can block mechanotransduction channels as well as ACh receptors in hair cells, they have no direct affect on OHC somatic motility.

Novel Plasmid-Mediated 16S rRNA m 1 A1408 Methyltransferase, NpmA, Found in a Clinically Isolated Escherichia coli Strain Resistant to Structurally Diverse Aminoglycosides

We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.

Molecular Determinants of Affinity for Aminoglycoside Binding to the Aminoglycoside Nucleotidyltransferase(2‘ ‘)-Ia, by

ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionMolecular Determinants of Affinity for Aminoglycoside Binding to the Aminoglycoside Nucleotidyltransferase(2‘ ‘)-Ia, byEdward Wright and Engin H. SerpersuCite this: Biochemistry 2007, 46, 35, 10236Publication Date (Web):August 7, 2007Publication History Published online7 August 2007Published inissue 1 September 2007https://doi.org/10.1021/bi701500gCopyright © 2007 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views178Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (7 KB) Get e-Alerts Get e-Alerts

Aminoglycosides versus bacteria – a description of the action, resistance mechanism, and nosocomial battleground

Since 1944, we have come a long way using aminoglycosides as antibiotics. Bacteria also have got them selected with hardier resistance mechanisms. Aminoglycosides are aminocyclitols that kill bacteria by inhibiting protein synthesis as they bind to the 16S rRNA and by disrupting the integrity of bacterial cell membrane. Aminoglycoside resistance mechanisms include: (a) the deactivation of aminoglycosides by N-acetylation, adenylylation or O-phosphorylation, (b) the reduction of the intracellular concentration of aminoglycosides by changes in outer membrane permeability, decreased inner membrane transport, active efflux, and drug trapping, (c) the alteration of the 30S ribosomal subunit target by mutation, and (d) methylation of the aminoglycoside binding site. There is an alarming increase in resistance outbreaks in hospital setting. Our review explores the molecular understanding of aminoglycoside action and resistance with an aim to minimize the spread of resistance.

New Insights into Aminoglycoside Antibiotics and Derivatives

In the post-genome era, RNA has emerged as a valid molecular target involved in many biochemical pathways such as cellular protein interactions. This includes transcription and translation, HIV-Rev and HIV-tat proteins binding to HIV RNA that encode HIV genetic information. The binding of natural and synthetic aminoglycosides to RNA or RNA-protein complexes has been a central focus of small molecules-RNA recognition events, since they bind selectively to a variety of RNA targets. The understanding of the contribution of shape-based and charge motifs involved in aminoglycosides-RNA complexes gives remarkable information of the translational miscoding process, thereby opening new insights for drug discovery. This review comprises relevant information about aminoglycosides antibiotics, including chemical structure, molecular mechanism, structure-activity relationships, recent advances in aminoglycoside mimetics over the last four years, resistance mechanisms and structure-toxicity relationships.

Use of 2-aminopurine as a fluorescent tool for characterizing antibiotic recognition of the bacterial rRNA A-site

Spectroscopic and calorimetric techniques have been employed to characterize the impact on an Escherichia coli rRNA A-site model oligonucleotide of incorporating the fluorescent base analog 2-aminopurine into the 1492- or the 1493-position, as well as the energetics and dynamics associated with recognition of this A-site model oligomer by aminoglycoside antibiotics. The results of these studies indicate that incorporation of 2AP into either the 1492- or the 1493-position does not perturb the structure or stability of the host RNA or its aminoglycoside binding affinity. In addition, the results also highlight drug-induced reduction in the mobilities of the bases at both the 1492- and 1493-positions as a potentially key determinant of bactericidal potency.

Abnormal organelles in cultured astrocytes are largely enhanced by streptomycin and intensively by gentamicin

The effects of two aminoglycoside antibiotics on cultured astrocyte organelles were investigated in rat, sheep, and human cultured astrocytes using transmission electron microscopy. Marked changes in mitochondrial shapes were observed in cultured or subcultured astrocytes obtained from three species, including humans. As well, new types of organelles were observed: (i) numerous concentric membranes forming vesicles, which were termed multilamellar vesicles; and (ii) many vesicles gathering into membranous structures, which were termed multivesicular myeloid bodies. The number of abnormalities increased proportionally with increasing concentrations of the two aminoglycosides (streptomycin and gentamicin). The incorporation of peroxidase or albumin–gold complex in the abnormal vesicles showed that the endolysosomal system was involved in the formation of these vesicles. Our results show that: abnormal organelles are present in cultured astrocytes; these abnormalities are enhanced by streptomycin and gentamicin; and gentamicin induces more abnormalities than streptomycin. The binding of aminoglycosides to membrane phospholipids may explain the formation of the observed abnormalities in rat, sheep, and human cultured astrocytes.

Binding of Aminoglycosidic Antibiotics to the Oligonucleotide A-Site Model and 30S Ribosomal Subunit: Poisson−Boltzmann Model, Thermal Denaturation, and Fluorescence Studies

The binding of paromomycin and similar antibiotics to the oligonucleotide A-site model and the small (30S) ribosomal subunit has been studied using continuum electrostatics methods. Crystallographic information from complexes of paromomycin, tobramycin, and Geneticin bound to an A-site oligonucleotide, and paromomycin and streptomycin complexed to the 30S subunit was used as a foundation to develop structures of similar antibiotics in the same ribosomal binding site. Relative binding free energies were calculated by combining the electrostatic contribution, which was obtained by solving the Poisson-Boltzmann equation, with a surface-area-dependent apolar term and contributions from conformational changes. These computed results showed good correlation with the experimental data resulting from fluorescence binding assays and thermal denaturation studies, demonstrating the ability of the Poisson-Boltzmann model to provide insight into the electrostatic mechanisms for aminoglycoside binding and direction for designing more effective antibiotics.