Monitoring tat peptide binding to TAR RNA by solid-state 31P-19F REDOR NMR

Abstract

Complexes of the HIV transactivation response element (TAR) RNA with the viral regulatory protein tat are of special interest due in particular to the plasticity of the RNA at this binding site and to the potential for therapeutic targeting of the interaction. We performed REDOR solid-state NMR experiments on lyophilized samples of a 29 nt HIV-1 TAR construct to measure conformational changes in the tat-binding site concomitant with binding of a short peptide comprising the residues of the tat basic binding domain. Peptide binding was observed to produce a nearly 4 A decrease in the separation between phosphorothioate and 2'F labels incorporated at A27 in the upper helix and U23 in the bulge, respectively, consistent with distance changes observed in previous solution NMR studies, and with models showing significant rearrangement in position of bulge residue U23 in the bound-form RNA. In addition to providing long-range constraints on free TAR and the TAR-tat complex, these results suggest that in RNAs known to undergo large deformations upon ligand binding, 31P-19F REDOR measurements can also serve as an assay for complex formation in solid-state samples. To our knowledge, these experiments provide the first example of a solid-state NMR distance measurement in an RNA-peptide complex.

Figure 1

REDOR pulse sequence used to measure ¹⁹F–³¹P dipolar couplings. Initial phosphorus magnetization is produced by cross polarization from ¹H to ³¹P. Application of a series of alternating rotor-synchronized fluorine and phosphorus 180° pulses dephases ³¹P transverse magnetization, resulting in a reduced ³¹P signal. The extent of dephasing observed is determined by the ¹⁹F–³¹P dipolar couplings. Comparison of this reduced signal with a reference signal obtained in the absence of the fluorine dephasing pulses allows the determination of the dipolar coupling, and thereby, direct extraction of the internuclear separation between ¹⁹F and ³¹P labeled sites.

Figure 2

Apical region of TAR RNA, showing 29-residue construct used in the present work. The hexanucleotide upper loop of wild-type TAR has been replaced with the stable GAAA tetraloop. Label positions within the RNA are shown as follows: ^2′FU indicates 2′-fluoro-2′-deoxyuridine (at bulge residue U23), and the dot between G26 and A27 denotes a phosphorothioate (pS) linkage.

Figure 3

(A and B) Conformational changes in TAR upon tat peptide binding. The phosphate backbone is represented by yellow ribbons, highlighting rearrangements of the bulge residues generated by peptide binding. Blue spheres denote ¹⁹F U23 and pS A27 label positions used in the present work. Illustrations and distances shown are adapted from solution NMR-derived models of the free and peptide-bound RNA (5,21). (A) Unbound conformation (PDB #1ANR, model one). (B) Bound conformation (PDB #1ARJ, model one.) Tat residue arg52, which provides key contacts with the RNA, is shown in orange. The RNA has straightened from the unbound conformation, residues C24 and U25 are looped out of the helix, and residue U23 has shifted to a new position adjacent to G26 and A27. (C and D) Rearrangements at TAR bulge binding site upon tat peptide binding. Blue spheres again denote ¹⁹F U23 and pS A27 label sites. (C) Close-up of unbound conformation, showing relative positions of U23, A27 and U38, and the inter-label separation monitored in the present study (PDB #1ANR, model one; ¹⁹F-pS inter-label separation is 12.3 Å in this model). (D) Close-up of bound conformation. Tat residue arg52 is shown in green. TAR accommodation of the tat peptide results in a significant repositioning of U23, drawing it into close proximity to A27 and U38, accompanied by a large decrease in separation between the ¹⁹F U23 and pS A27 label positions (PDB #1ARJ, model one.) Following binding, the ¹⁹F–pS distance has decreased to 5.3 Å in the model shown.

Figure 4

³¹P MAS spectra of the unbound (A) and bound (B) TAR RNA. Plots were generated without linebroadening and show reference (S₀) spectra corresponding to REDOR S/S₀ points acquired at 1.23 ms. In each spectrum, the phosphorothioate peak (pS A27) monitored to measure REDOR dephasing is denoted by ‘pS’; the remaining peaks are isotropic and spinning sideband peaks owing to unmodified backbone phosphates.

Figure 5

REDOR dephasing curves for unbound TAR RNA and TAR–tat peptide complex. Diamonds mark data for the unbound RNA, and triangles for the complex. Solid lines represent simulated dephasing corresponding to 10.3 and 6.6 Å, respectively. All simulations used SIMPSON, as described in the text.