Native mass spectrometry reveals the initial binding events of HIV-1 rev to RRE stem II RNA

Abstract

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.

Mass Spectrometry of Nucleic Acid Noncovalent Complexes

“…As electrostatic interactions between the peptides and oligonucleotides are strong enough to survive in the gas phase, , native top-down mass spectrometry of these complexes can be used to map the binding site. Complexes of HIV-1 TAR (transactivation responsive) RNA and a basic arginine-rich peptide (H 2 N-GRKK­RRQR­RRPP-NH 2 ) were studied by CID .…”

“…Time-dependent MS data showed 1:2 and 1:1 stoichiometries of TAR-tat complexes, shown in Figure . Native mass spectrometry revealed the binding stoichiometry and binding mechanism of RRE stem IIB RNA with rev ARM peptides . ESI-MS showed that stem IIB RNA formed complexes with one or two ARM peptides and full length RRE stem IIB RNA binds with one or five rev ARM peptides.…”

“…Native mass spectrometry revealed the binding stoichiometry and binding mechanism of RRE stem IIB RNA with rev ARM peptides. 1166 ESI-MS showed that stem IIB RNA formed complexes with one or two ARM peptides and full length RRE stem IIB RNA binds with one or five rev ARM peptides. The binding site mapping was done by CID.…”

Step toward Probing the Nonannular Belt of Membrane Proteins

“…Native mass spectrometry (MS) is a powerful biophysical technique for studying bio-macromolecular assemblies and their interactions with molecules. − Under carefully tuned conditions, native MS preserves noncovalent interactions in the gas phase , and provides insight into individual ligand binding events and binding stoichiometry. For this reason, native MS can be utilized to elucidate the interactions between biomolecules and small molecules, such as for proteins interacting with lipids, drugs, nucleotides, and peptides. − Native MS has also advanced to determine binding constants and thermodynamics of protein–lipid interactions and allostery between protein–lipid interactions and small molecules, such as other lipids, , and drugs. , …”

Characterization of the T4 gp32–ssDNA complex by native, cross-linking, and ultraviolet photodissociation mass spectrometry

“…The use of MS/MS for structural interrogation of oligonucleotidecontaining complexes has emerged as useful for a few studies focused on ligand localization and conformational changes. 22,26,28,31,34,45 However, in the case of non-covalent complexes, collisional activation methods, although providing some sequence information, do not afford extensive information related to ligand localization and overall complex structure. To combat these decits, alternative activation methods that are more suitable for the analysis of non-covalent macromolecular complexes have been developed, including surfaceinduced dissociation (SID), 46 electron activation methods, 47 and ultraviolet photodissociation (UVPD).…”