Determinants of aminoglycoside-binding specificity for rRNA by using mass spectrometry

Abstract

We have developed methods for studying the interactions between small molecules and RNA and have applied them to characterize the binding of three classes of aminoglycoside antibiotics to ribosomal RNA subdomains. High-resolution MS was used to quantitatively identify the noncovalent binding interactions between mixtures of aminoglycosides and multiple RNA targets simultaneously. Signal overlap among RNA targets was avoided by the addition of neutral mass tags that direct each RNA target to a unique region of the spectrum. In addition to determining binding affinities, the locations of the binding sites on the RNAs were identified from a protection pattern generated by fragmenting the aminoglycoside͞RNA complex. Specific complexes were observed for the prokaryotic rRNA A-site subdomain with ribostamycin, paromomycin, and lividomycin, whereas apramycin preferentially formed a complex with the eukaryotic subdomain. We show that differences in binding between paromomycin and ribostamycin can be probed by using an MS-MS protection assay. We have introduced specific base substitutions in the RNA models and have measured their impact on binding affinity and selectivity. The binding of apramycin to the prokaryotic subdomain strongly depends on the identity of position 1408, as evidenced by the selective increase in affinity for an A1408G mutant. An A1409-G1491 mismatch pair in the prokaryotic subdomain enhanced the binding of tobramycin and bekanamycin. These observations demonstrate the power of MS-based methods to provide molecular insights into small molecule͞RNA interactions useful in the design of selective new antimicrobial drugs.The aminoglycoside antibiotics are potent inhibitors of protein synthesis and RNA splicing in vitro and in vivo (1-6). As the first class of compounds known to bind specifically to subdomains of larger RNA sequences, they are useful for understanding the design principles required to produce new classes of therapeutic agents to bind RNAs (7, 8). The best characterized aminoglycoside binding site is the decoding region of the small ribosomal subunit. The structures of paromomycin and gentamicin C1a complexes with a model RNA subdomain have been determined by using NMR (9-12). These aminoglycosides make hydrogen-bonding, electrostatic, and hydrophobic contacts that contribute to their high binding affinity for the RNA target.We have developed methods that facilitate quantitative analysis of the binding characteristics of multiple aminoglycosides with a series of structural variants of the RNA to decipher further the molecular details of how different classes of aminoglycosides can discriminate among structurally related RNAs. We show that electrospray ionization MS (ESI-MS) has unique advantages for rapid characterization of RNA binding with aminoglycosides or any chemical or biological substrate. Mixtures of compounds can be screened in parallel with a high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer, because the exact molecular mass of each lig...

Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity

“…Model oligonucleotides designed to mimic the drug-binding site have been used to investigate various aspects of aminoglycoside-ribosome interaction (18)(19)(20)(21)(22). However, conclusions derived from the study of model A-site oligonucleotides are compromised by several findings: (i) in contrast to drug susceptibility of complete ribosomes, binding affinities of aminoglycosides to prokaryotic decoding region constructs are not very sensitive to mutations within the RNA-binding region (23); (ii) in vivo drug susceptibilities of mutant ribosomes and in vitro binding affinities using variants of model A-site oligonucleotides may or may not correlate (24)(25)(26); (iii) the exquisite specificity of aminoglycosides for the prokaryotic as opposed to the eukaryotic cytosolic ribosome contrasts with the observation that these drugs bind to eukaryotic decoding-site constructs with approximately the same affinity as found for their prokaryotic counterpart (23,24); and (iv) while there is evidence that mitochondrial ribosomes are susceptible to aminoglycosides (13,27), oligonucleotides mimicking the mitochondrial A site do not bind aminoglycosides to any significant extent (24,28).…”

Mapping noncovalent ligand binding to stemloop domains of the HIV-1 packaging signal by tandem mass spectrometry

“…Targeted fragmentation of the phosphodiester backbone has been previously utilized to screen members of combinatorial libraries for their ability to bind specific targets [38,57]. Based on the observation that RNA and 2=-O-methyl RNA undergo fragmentation 5 to 10 times less efficiently than DNA [58], a chimeric 2=-O-methylribonucleotide corresponding to the bacterial rRNA A-site was synthesized with deoxyadenine mutations located in selected sequence positions to induce facile gas-phase cleavage.…”

A eole for the MS analysis of nucleic acids in the post-genomics age

“…Unlike these techniques, MS is capable of resolving any free/bound species at equilibrium in solution, even when such species possess very similar spectroscopic characteristics. With proper experimental design and data treatment, their respective signal intensities can be employed to obtain relative [121,122] and absolute [123][124][125][126][127][128] dissociation constants (K d 's) in solution, matching those afforded by established methods [129]. In this direction, competitive binding experiments in which multiple ligands are mixed simultaneously with the substrate of interest have proven very effective in providing relative scales of binding affinities based on the aspect ratio and distribution of the detected complexes [130 -133].…”

“…This observation has promoted strategies for achieving the characterization of specific binding sites onto target nucleic acid structures, which are revealed by recognizable gaps in the detected ion series [134,171,172]. Conversely, the same protection effects can be employed to screen libraries of small molecule ligands for their ability to bind to desired structural motifs, which is evaluated from their power to induce sitedirected inhibition of nucleic acid fragmentation [123,173]. In the absence of covalent fragmentation, the order by which bound subunits dissociate as discrete products may reveal their spatial situation within the complex of interest.…”