Nuclear export complexes composed of rev response element (RRE) ribonucleic acid (RNA) and multiple molecules of rev protein are promising targets for the development of therapeutic strategies against human immunodeficiency virus type 1 (HIV-1), but their assembly remains poorly understood. Using native mass spectrometry, we show here that rev initially binds to the upper stem of RRE IIB, from where it is relayed to binding sites that allow for rev dimerization. The newly discovered binding region implies initial rev recognition by nucleotides that are not part of the internal loop of RRE stem IIB RNA, which was previously identified as the preferred binding region. Our study highlights the unique capability of native mass spectrometry to separately study the binding interfaces of RNA/protein complexes of different stoichiometry, and provides a detailed understanding of the mechanism of RRE/rev association with implications for the rational design of potential drugs against HIV-1 infection.
Interactions of ribonucleic
acids (RNA) with basic ligands such
as proteins or aminoglycosides play a key role in fundamental biological
processes. Native top-down mass spectrometry (MS) has recently been
extended to binding site mapping of RNA–ligand interactions
by collisionally activated dissociation, without the need for laborious
sample preparation procedures. The technique relies on the preservation
of noncovalent interactions at energies that are sufficiently high
to cause RNA backbone cleavage. In this study, we address the question
of how many and what types of noncovalent interactions allow for binding
site mapping by top-down MS. We show that proton transfer from protonated
ligand to deprotonated RNA within salt bridges initiates loss of the
ligand, but that proton transfer becomes energetically unfavorable
in the presence of additional hydrogen bonds such that the noncovalent
interactions remain stronger than the covalent RNA backbone bonds.
Interactions of ribonucleic acid (RNA) with guanidine and guanidine derivatives are important features in RNA–protein and RNA–drug binding. Here we have investigated noncovalently bound complexes of an 8‐nucleotide RNA and six different ligands, all of which have a guanidinium moiety, by using electrospray ionization (ESI) and collisionally activated dissociation (CAD) mass spectrometry (MS). The order of complex stability correlated almost linearly with the number of ligand atoms that can potentially be involved in hydrogen‐bond or salt‐bridge interactions with the RNA, but not with the proton affinity of the ligands. However, ligand dissociation of the complex ions in CAD was generally accompanied by proton transfer from ligand to RNA, which indicated conversion of salt‐bridge into hydrogen‐bond interactions. The relative stabilities and dissociation pathways of [RNA+m L−n H]n− complexes with different stoichiometries (m=1–5) and net charge (n= 2–5) revealed both specific and unspecific ligand binding to the RNA.
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