Monitoring aminoglycoside-induced conformational changes in 16S rRNA through acrylamide quenching

Chao, Pei Wen; Chow, Christine S.

doi:10.1016/j.bmc.2007.03.025

Cited by 22 publications

(29 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The enhanced exposure towards RNase A cleavage is suggestive of possible base flipping of the two adenine residues. Such an induced conformational change upon binding of aminoglycosides to the A-site RNA has been observed previously in model RNAs (14, 47–49) and in X-ray crystal structures and molecular modeling studies of the complete ribosome (7, 11, 50, 51). The binding mode of HPVHHYQ-NH 2 , with respect to base flipping, may be similar to the antibiotics such as paromomycin, but with a reduced electrostatic component of binding.…”

Section: Discussionsupporting

confidence: 75%

Selection of Peptides That Target the Aminoacyl-tRNA Site of Bacterial 16S Ribosomal RNA

Duc

Klosi

et al. 2009

Biochemistry

Self Cite

View full text Add to dashboard Cite

For almost five decades, antibiotics have been used successfully to control infectious diseases caused by bacterial pathogens. More recently, however, two-thirds of bacterial pathogens exhibit resistance and are continually evolving new resistance mechanisms against almost every clinically used antibiotic. Novel efforts are required for the development of new drugs or drug leads to combat these infectious diseases. A number of antibiotics target the bacterial aminoacyl-tRNA site (A site) of 16S ribosomal RNA (rRNA). Mutations in the A-site region are known to cause antibiotic resistance. In this study, a bacterial (E. coli) A-site rRNA model was chosen as a target to screen for peptide binders. Two heptapeptides, HPVHHYQ and LPLTPLP, were selected through M13 phage display. Both peptides display selective binding to the A-site 16S rRNA with on-bead fluorescence assays. Dissociation constants (K d s) of the amidated peptide HPVHHYQ-NH 2 to various A-site RNA constructs were determined by using enzymatic footprinting, electrospray ionization mass spectrometry (ESI-MS), and isothermal titration calorimetry (ITC) under a variety of buffer and solution conditions. HPVHHYQ-NH 2 exhibits moderate affinity for the A-site RNA, with an average K d value of 16 μM. In addition, enzymatic footprinting assays and competition ESI-MS with a known A-site binder (paromomycin) revealed that peptide binding occurs near the asymmetric bulge at positions U1495 and G1494 and leads to increased exposure of residues A1492 and A1493.It has been half a century since the first antibiotic penicillin was discovered and successfully applied to the treatment of infectious diseases; however, infectious diseases still remain the third-leading cause of death in the United States and the second-leading cause of death worldwide (1). Widespread and intensive use of antibiotics in hospitals and agriculture has led to significant levels of resistance (2,3). The emergence of antibiotic resistance, especially multidrug resistance, is a growing threat to human health (4). Therefore, a significant driving force exists for the development of new antimicrobial agents, especially those to combat multidrug-resistant pathogens.The majority of antibiotics used in the clinic target protein synthesis, which occurs at the core of the ribosome (5). High-resolution X-ray crystal structures of rRNA fragments, 30S and 50S subunits, and whole ribosomes, either free or complexed with antibiotics, have provided important information that will help in the design of new anti-infective compounds (6-10). † This work was supported by NIH grant AI061192. SUPPORTING INFORMATION AVAILABLEFigures S1-S4 show MALDI-TOF characterization of HPVHHYQ-NH 2 , ESI-MS data (salt dependence), and binding curve for RNase footprinting data (A1493). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 September 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH...

show abstract

Section: Discussionsupporting

confidence: 75%

Selection of Peptides That Target the Aminoacyl-tRNA Site of Bacterial 16S Ribosomal RNA

Duc

Klosi

et al. 2009

Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Their electrostatic interactions with the negatively charged RNA provide a major contribution to the binding free energy26,51–53. It was suggested that, upon binding, aminoglycosides expel positive ions that natively occupy the RNA bulge12,54,55. In order to check if there is any localized ion density in the binding site in the absence of paromomycin, we analyzed an average distribution of sodium ions in the simulations without the antibiotic.…”

Section: Resultsmentioning

confidence: 99%

“…Upon binding of paromomycin, these adenines protrude from the 16S rRNA helix and make space for the antibiotic3,10–12 — we will refer to this configuration as the flipped-out or extra-helical state. In contrast, when the antibiotic is absent, these two adenines tend to be hidden inside the RNA helix — in a flipped-in or intra-helical state13,14.…”

Section: Introductionmentioning

confidence: 99%

Molecular Dynamics Study of the Ribosomal A-Site

2008

View full text Add to dashboard Cite

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 obtained configurations of the A-site that were also observed in the NMR and crystal structures. Our studies show the differences in the internal mobility of the A-site, as well as in ion and water density distributions inside the binding cleft, between the prokaryotic and mutated RNA. We also compare the performance of two force field parameters for RNA -Amber and Charmm.

show abstract

“…Fluorescent A-site constructs, which contain emissive and responsive nucleoside analogues, such as 2-aminopurine at positions 1492 or 1493, have shown great promise 28–31. Their fluorescence response is, however, antibiotic-dependent 28,32. To overcome this drawback and devise a robust analysis and discovery platform for A-site binders, we have envisioned an approach where detection of antibiotic binding is not dependent on changes in the environment of a single fluorophore, but rather on the interaction between two chromophores acting as a Förster Resonance Energy Transfer (FRET) pair (Figure 1b).…”

Section: Introductionmentioning

confidence: 99%

FRET Enabled Real Time Detection of RNA-Small Molecule Binding

2009

View full text Add to dashboard Cite

A robust analysis and discovery platform for antibiotics targeting the bacterial rRNA A-site has been developed by incorporating a new emissive U surrogate into the RNA and labeling the aminoglycosides with an appropriate fluorescence acceptor. Specifically, a 5-methoxyquinazoline-2,4(1H,3H)-dione-based emissive uracil analogue was identified to be an ideal donor for 7-diethylaminocoumarin-3-carboxylic acid. This donor/acceptor pair displays a critical Förster radius (R0) of 27 Å, a value suitable for an A-site-aminoglycoside assembly. Titrating the coumarin labeled aminoglycosides into the emissive A-site construct, labeled at position U1406, shows a decrease in donor emission (at 395 nm) and concurrent increase of the acceptor emission (at 473 nm). Titration curves, obtained by fitting the donor’s emission quenching or the augmentation of the acceptor’s sensitized emission, faithfully generate EC50 values. Titration of unlabeled ligands into the preformed FRET complex showed a continuous increase of the donor emission, with a concurrent decrease of the acceptor emission, yielding valuable data regarding competitive displacement of aminoglycosides by A-site binders. Detection of antibiotic binding is therefore not dependent on changes in the environment of a single fluorophore, but rather on the responsive interaction between two chromophores acting as a FRET pair, facilitating the determination of direct binding and competitive displacement events with FRET accuracy.

show abstract

Monitoring aminoglycoside-induced conformational changes in 16S rRNA through acrylamide quenching

Cited by 22 publications

References 36 publications

Selection of Peptides That Target the Aminoacyl-tRNA Site of Bacterial 16S Ribosomal RNA

Selection of Peptides That Target the Aminoacyl-tRNA Site of Bacterial 16S Ribosomal RNA

Molecular Dynamics Study of the Ribosomal A-Site

FRET Enabled Real Time Detection of RNA-Small Molecule Binding

Contact Info

Product

Resources

About