Native Top‐Down Mass Spectrometry of TAR RNA in Complexes with a Wild‐Type tat Peptide for Binding Site Mapping

Abstract

Ribonucleic acids (RNA) frequently associate with proteins in many biological processes to form more or less stable complex structures.T he characterization of RNAprotein complex structures and binding interfaces by nuclear magnetic resonance (NMR) spectroscopy, X-rayc rystallography,orstrategies based on chemical crosslinking,however,can be quite challenging.H erein, we have explored the use of an alternative method, native top-down mass spectrometry (MS), for probing of complex stoichiometry and protein binding sites at the single-residue level of RNA. Our data show that the electrostatic interactions between HIV-1 TARR NA and ap eptide comprising the arginine-richb inding region of tat protein are sufficiently strong in the gas phase to survive phosphodiester backbone cleavage of RNAb yc ollisionally activated dissociation (CAD), thus allowing its use for probing tat binding sites in TARRNA by top-down MS.Moreover,the MS data reveal time-dependent 1:2a nd 1:1s toichiometries of the TAR-tat complexes and suggest structural rearrangements of TARRNA induced by binding of tat peptide.Interactions between ribonucleic acids (RNA) and proteins are central to many fundamental biological processes,including gene expression and infection by RNAv iruses.F or athorough understanding of such interactions,RNA-protein complexes are commonly investigated by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, both of which require relatively large quantities of sample material. Moreover,N MR data interpretation can be complicated by unfavorable conformational dynamics, [1] and crystallography can become impossible if ac omplex fails to crystallize properly.S trategies based on (photo)chemical crosslinking [2] have the advantage that they can be performed in vivo [3] but can variously suffer from low crosslinking yields, different crosslinking reactivity of different residues,o rt he formation of intramolecular instead of intermolecular crosslinks.[4] Moreover,c rosslinking reagents generally target specific functional groups such as amines or thiols that may not be present in ab inding region, and efficient crosslinking can require pH values that may not be compatible with RNAprotein complex stability.[5] Although all of the above techniques can provide highly important structural data, each of them requires laborious sample preparation procedures,t hat is,c rystallization, the introduction of heavy isotopes,o ro ptimization of the reaction conditions for the formation of intermolecular crosslinks.As an alternative to these methods,w ee xplore the potential of native top-down mass spectrometry (MS) using electrospray ionization (ESI) [6] fort he characterization of RNA-protein interactions.P revious studies showed that native ESI can produce gaseous RNA-protein or RNAligand complexes, [7] and in top-down MS experiments using collisionally activated dissociation (CAD), Loo [8] and Fabris [7f] observed cleavage of covalent mononucleotide phosphate and RNAp hosphodiester bonds,r espectively,r ather...

Structural Characterization of a Thrombin-Aptamer Complex by High Resolution Native Top-Down Mass Spectrometry

“…Tandem MS of protein-nucleic acid complexes has recently showed progress to address complexes of larger DNA/RNA molecules, beyond the earlier work with single nucleotides [23, 49]. The sites of peptide binding to a 31-nucleotide sequence of TAR RNA were recently elucidated by native top-down MS [50]. As larger protein-nucleic acid complexes are probed by native top-down MS, better models will be needed to help us better understand the dissociation process of complexes composed of large, highly negatively charged molecules bound to proteins [51].…”

Higher-order structural characterisation of native proteins and complexes by top-down mass spectrometry

“…165 Schneeberger and Breuker have used CID to determine the binding site of RNA on proteins, again taking advantage of the strengthening in the gas phase of electrostatic interactions to the point where they are occasionally able to survive backbone fragmentation. 166 Recently, O'Connor and coworkers have shown that ECD fragmentation of oligomers of the amyloidogenic peptide amylin (implicated in type 2 diabetes) can provide information on the binding interface between monomers (see Fig. 6).…”

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

“…To gain insight into the biochemistry of retroviruses and further antiviral drug development, atomic-level details of RNA-protein complex structures and their binding interfaces can be studied by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography. An emerging alternative that can complement these techniques, especially when RNAprotein complexes show unfavorable conformational dynamics that complicate NMR data interpretation 2,3 or fail to crystallize properly 4,5 , is native top-down mass spectrometry (MS) 6 . We have recently demonstrated that native electrospray ionization (ESI) [7][8][9][10][11][12] provides time-resolved, stoichiometric information on complexes of transactivation responsive (TAR) RNA and transactivating (tat) peptide from human immunodeficiency virus type 1 (HIV-1), and that collisionally activated dissociation (CAD) can reveal tat binding sites of TAR RNA at the single-nucleotide level 6 .…”

“…An emerging alternative that can complement these techniques, especially when RNAprotein complexes show unfavorable conformational dynamics that complicate NMR data interpretation 2,3 or fail to crystallize properly 4,5 , is native top-down mass spectrometry (MS) 6 . We have recently demonstrated that native electrospray ionization (ESI) [7][8][9][10][11][12] provides time-resolved, stoichiometric information on complexes of transactivation responsive (TAR) RNA and transactivating (tat) peptide from human immunodeficiency virus type 1 (HIV-1), and that collisionally activated dissociation (CAD) can reveal tat binding sites of TAR RNA at the single-nucleotide level 6 . This native top-down MS approach relies on the preservation of electrostatic interactions between protonated peptides and deprotonated RNA at energies that are sufficiently high for phosphodiester backbone bond cleavage 6 , for which as few as two arginine residues are sufficient 13 .…”