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

Abstract

The binding modes and structural determinants of the noncovalent complexes formed by aminoglycoside antibiotics with conserved domains of the HIV-1 packaging signal (⌿-RNA) were investigated using electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). The location of the aminoglycoside binding sites on the different stemloop structures was revealed by characteristic coverage gaps in the ion series obtained by sustained off-resonance irradiation collision induced dissociation (SORI-CID) of the antibiotic-RNA assemblies. The site positions were confirmed using mutants that eliminated salient structural features of the ⌿-RNA domains. The effects of the mutations on the binding properties of the different substrates served to validate the position of the aminoglycoside site on the wild-type structures. Additional information was provided by docking experiments performed on the different aminoglycoside-stemloop complexes. The results have shown that, in the absence of features disrupting the regular A-helix of the double-stranded stem, aminoglycosides tend to bind in an area situated between the upper stem and the loop regions, as demonstrated for stemloop SL3. The presence of a tandem wobbles motif in SL4 modifies the regular geometry of the upper stem, which does not affect the general site location, but greatly increases its solution binding affinity compared with SL3. The platform motif in SL2 locates the binding site in the stem midsection and confers upon this stemloop an intermediate affinity toward aminoglycosides. In SL3 and SL4, the extensive overlap of the antibiotic site with the region used to bind the nucleocapsid (NC) protein provides the basis for a competition mechanism that could explain the aminoglycoside inhibition of the NC·SL3 and NC·SL4 assemblies. In contrast, the minimal overlap between the aminoglycoside and the NC sites in SL2 accounts for the absence of inhibition of the NC·SL2 complex. . The relatively low mutation frequency of this ϳ120 nt stretch of genomic RNA makes the packaging signal a very promising target for the development of new therapeutic strategies to contend with the rapid emergence of drug resistant strains [7,8]. For this reason, steps have been made toward the identification of possible inhibitors that may bind specific ⌿-RNA structures and disrupt their normal functions [9 -11]. In a systematic investigation of classic nucleic acid ligands, we have recently shown that aminoglycosidic antibiotics are not only capable of binding individual domains of ⌿-RNA, but can also inhibit their interactions with NC in a structure-specific fashion [12]. Initial tests to locate the actual binding sites on the different RNA substrates, which is crucial to explain the mechanism of preferential inhibition, were based on the analysis of mutant-ligand complexes performed by electrospray ionization (ESI) [13,14] and Fourier transform mass spectrometry (FTMS) [15,16].The favorable energetics characteristic of ESI enable the analysis of relatively labile noncovalent complex...

Higher-order structure of nucleic acids in the gas phase: Top-down analysis of base-pairing interactions

“…In this direction, we have employed sustained off-resonance irradiation (SORI) 24 CID to characterize the unique structural features exhibited by DNA-RNA hybrid duplexes 25, 26 and RNA stemloop structures 27, 28 involved in viral replication. The observation that backbone fragmentation could be inhibited by the contact of non-covalent ligands 27 prompted us to propose the utilization of classic nucleic acid ligands as non-covalent structural probes.…”

Top-down mass spectrometry for sequencing of larger (up to 61 nt) RNA by CAD and EDD

“…Topdown MS has been applied to study RNA modified by structural probes [20], for characterization of conserved domains of the HIV-1 packaging signal RNA [21], for investigating aptamer/ligand complexes [22], and binding of antibiotics to ribosomal RNA subdomains [23]. However, sequencing by top-down mass spectrometry of RNA Ͼ 20 nt has been demonstrated only recently, as discussed below.…”

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

“…Under suitable activation conditions, the binding of selected ligands to RNA substrates can prevent underlying nucleotides from undergoing the typical backbone fragmentation that produces the characteristic ion series employed in MS/MS sequencing. 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].…”