Nathan A. Hagan

Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides

The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers. It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replication cycle as well. We previously reported that NC binds with high-affinity to the repeating sequence d(TG)n. We have now analyzed the interaction between NC and d(TG)4 in considerable detail, using surface plasmon resonance (SPR), tryptophan fluorescence quenching (TFQ), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC) and electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). Our results show that the interactions between these two molecules are surprisngly complex: while the Kd for binding of a single d(TG)4 molecule to NC is only ∼5 nM in 150 mM NaCl, a single NC molecule is capable of interacting with more than one d(TG)4 molecule, and conversely, more than one NC molecule can bind to a single d(TG)4 molecule. The strengths of these additional binding reactions are quantitated. The implications of this multivalency for the functions of NC in virus replication are discussed.

Inhibitory effects of archetypical nucleic acid ligands on the interactions of HIV-1 nucleocapsid protein with elements of Ψ-RNA

Disrupting the interactions between human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein and structural elements of the packaging signal (Ψ-RNA) could constitute an ideal strategy to inhibit the functions of this region of the genome leader in the virus life cycle. We have employed electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS) to assess the ability of a series of nucleic acid ligands to bind selected structures of Ψ-RNA and inhibit their specific interactions with NC in vitro. We found that the majority of the ligands included in the study were able to form stable non-covalent complexes with stem–loop 2, 3 and 4 (SL2–4), consistent with their characteristic nucleic acid binding modes. However, only aminoglycosidic antibiotics were capable of dissociating preformed NC•SL3 and NC•SL4 complexes, but not NC•SL2. The apparent specificity of these inhibitory effects is closely dependent on distinctive structural features of the different NC•RNA complexes. The trends observed for the IC50 values correlate very well with those provided by the ligand binding affinities and the dissociation constants of target NC•RNA complexes. This systematic investigation of archetypical nucleic acid ligands provides a valid framework to support the design of novel ligand inhibitors for HIV-1 treatment.

Noncovalent probes for the investigation of structure and dynamics of protein-nucleic acid assemblies: the case of NC-mediated dimerization of genomic RNA in HIV-1.

The nature of specific RNA-RNA and protein-RNA interactions involved in the process of genome dimerization and isomerization in HIV-1, which is mediated in vitro by stemloop 1 (SL1) of the packaging signal and by the nucleocapsid (NC) domain of the viral Gag polyprotein, was investigated by using archetypical nucleic acid ligands as noncovalent probes. Small-molecule ligands make contact with their target substrates through complex combinations of H-bonds, salt bridges, and hydrophobic interactions. Therefore, their binding patterns assessed by electrospray ionization mass spectrometry can provide valuable insights into the factors determining specific recognition between species involved in biopolymer assemblies. In the case of SL1, dimerization and isomerization create unique structural features capable of sustaining stable interactions with classic nucleic acid ligands. The binding modes exhibited by intercalators and minor groove binders were adversely affected by the significant distortion of the duplex formed by palindrome annealing in the kissing-loop (KL) dimer, whereas the modes observed for the corresponding extended duplex (ED) confirmed a more regular helical structure. Consistent with the ability to establish electrostatic interactions with highly negative pockets typical of helix anomalies, polycationic aminoglycosides bound to the stem-bulge motif conserved in all SL1 conformers, to the unpaired nucleotides located at the hinge between kissing hairpins in KL, and to the exposed bases flanking the palindrome duplex in ED. The patterns afforded by intercalators and minor groove binders did not display detectable variations when the corresponding NC-SL1 complexes were submitted to probing. In contrast, aminoglycosides displayed the ability to compete with the protein for overlapping sites, producing opposite effects on the isomerization process. Indeed, displacing NC from the stem-bulges of the KL dimer induced inhibition of stem melting and decreased the efficiency of isomerization. Competition for the hinge region, instead, eliminated the NC stabilization of a grip motif formed by nucleobases of opposite strands, thus facilitating the strand-exchange required for isomerization. These noncovalent probes provided further evidence that the structural context of the actual binding sites has significant influence on the chaperone activities of NC, which should be taken in account when developing potential drug candidates aimed at disrupting genome dimerization and isomerization in HIV-1.

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

The binding modes and structural determinants of the noncovalent complexes formed by aminoglycoside antibiotics with conserved domains of the HIV-1 packaging signal (Ψ-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 Ψ-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.

Functional investigations of retroviral protein-ribonucleic acid complexes by nanospray Fourier transform ion cyclotron resonance mass spectrometry

Nanospray-FT-ICR has been employed to investigate the processes of genome dimerization, selection, and packaging in human immunodifficiency virus type 1, which are mediated by specific interactions between the nucleocapsid protein (NC) and the structural elements formed by the genomes packaging signal [ψ-ribonucleic acid (RNA)]. This analytical platform allowed for the unambiguous characterization of all the non-covalent complexes formed in vitro by simultaneous RNA–RNA and protein–RNA binding equilibria. Competitive binding experiments involving the isolated RNA elements were completed to evaluate their ability to sustain specific protein interactions. In similar fashion, ad hoc RNA mutants were used to locate two distinct binding sites on the apical loop and stem-bulge of the monomeric stemloop 1 (SL1) domain, which is responsible for initiating the dimerization process. The stem-bulge motifs provided viable binding sites in both the kissing-loop (KL) and the extended duplex forms of dimeric SL1, whereas the latter included additional sites corresponding to the A-bulge motifs that flank the annealed palindromes. A cross-linking approach using pre-derivatized, photo-cross-linkable NC demonstrated that the SL3 domain was the preferred site for protein binding in the context of full-length ψ-RNA. This concerted strategy is expected to provide new valuable insight into the effects induced by the global folding of ψ-RNA on its ability to interact with the NC protein during genome dimerization, selection and packaging.