Characterizing complex dynamics in the transactivation response element apical loop and motional correlations with the bulge by NMR, molecular dynamics, and mutagenesis

Abstract

The HIV-1 transactivation response element (TAR) RNA binds a variety of proteins and is a target for developing anti-HIV therapies. TAR has two primary binding sites: a UCU bulge and a CUGGGA apical loop. We used NMR residual dipolar couplings, carbon spin relaxation (R(1) and R(2)), and relaxation dispersion (R(1rho)) in conjunction with molecular dynamics and mutagenesis to characterize the dynamics of the TAR apical loop and investigate previously proposed long-range interactions with the distant bulge. Replacement of the wild-type apical loop with a UUCG loop did not significantly affect the structural dynamics at the bulge, indicating that the apical loop and the bulge act largely as independent dynamical recognition centers. The apical loop undergoes complex dynamics at multiple timescales that are likely important for adaptive recognition: U31 and G33 undergo limited motions, G32 is highly flexible at picosecond-nanosecond timescales, and G34 and C30 form a dynamic Watson-Crick basepair in which G34 and A35 undergo a slow (approximately 30 mus) likely concerted looping in and out motion, with A35 also undergoing large amplitude motions at picosecond-nanosecond timescales. Our study highlights the power of combining NMR, molecular dynamics, and mutagenesis in characterizing RNA dynamics.

FIGURE 1

NMR chemical shift comparison of wtTAR and TAR_m. (a) Secondary structures of wtTAR and TAR_m. Residues with the largest chemical shift perturbations (>0.25 ppm) between wtTAR and TAR_m are highlighted on the wtTAR secondary structure using solid black symbols. Apical loop residues that undergo significant chemical shift perturbations upon ARG binding are highlighted in open black symbols. See inset in c for symbol key. (b) 2D ¹H-¹³C HSQC spectra of wtTAR (red) overlaid on corresponding spectra of TAR_m (blue). Asterisks and colored labels denote resonances that are different between wtTAR and TAR_m due to the loop mutation. (c) Chemical shift differences ( where and are the changes in proton and carbon/nitrogen chemical shift and α is the ratio of the H and C/N gyromagnetic ratio) between wtTAR and TAR_m.

FIGURE 2

RDC-based comparison of the wtTAR and TAR_m conformational dynamics. (a) Correlation plot between measured and back-calculated wtTAR RDCs when each stem order tensor is independently fit to an idealized A-form geometry. Domain II is shown in gray, and domain I is shown in black, with A22 and U40 represented by open black circles. (b) The values for the interhelical bend (θ), twist angle (ξ), and the interhelical mobility (ϑ_int) determined for wtTAR (black) and TAR_m (gray). Experimental errors are shown.

FIGURE 3

Spin relaxation-based comparison of the E-wtTAR and E-TAR_m conformational dynamics. (a) Elongated construct of wtTAR in which XY are unlabeled CG (E-GC-wtTAR) and UA (and E-AU-wtTAR) residues as previously described (29). Residues that undergo significant chemical shift perturbations upon elongation are highlighted on the secondary structure using black solid symbols. See inset in Fig. 1 d for symbol key. (b) 2D HSQC spectra of wtTAR (red) overlaid on corresponding spectra of E-AU-wtTAR and E-GC-wtTAR (black). Blue labels denote loop resonances that shift upon elongation. (c) Correlation plot between the E-wtTAR and E-TAR_m normalized intensities. Shown is the correlation coefficient (R). (d) Normalized resonance intensities (peak heights) measured from 2D HSQC spectra of E-wtTAR. See inset for key. (e) Correlation plot between nucleobase carbon values measured in nonelongated wtTAR and TAR_m. Values for domain I, bulge, and domain II are shown in red, orange, and green circles, respectively.

FIGURE 4

Dynamics of the wtTAR loop. Shown are the (a) normalized intensities for E-wtTAR (solid symbols) and values for nonelongated wtTAR (open symbols) and (b) one-bond C-H RDCs measured in the wtTAR loop (see inset for key). (c) Carbon R₂ as a function of field strength (ω_eff) for residues G34 (C8) and A35 (C8). Note that R₂ = R_2,int + R_ex = R_1ρ/sin²θ − R₁tan²θ. (d) Two snapshots from the 65 ns MD simulation of wtTAR illustrating the looping in and out of A35. (e) Schematic diagram of the observed wtTAR apical loop dynamics. Nucleobases, ribose moieties, and phosphate groups are represented by large rectangles, black pentagons, and solid black circles, respectively. Smaller black rectangles denote base-base stacking. Gray circles indicate hydrogen bonding between bases. The open gray rectangle indicates transient base-base stacking. The open gray circle indicates a transient hydrogen bond across the loop. Fast motions are indicated by solid arrows. The looping in and out of A35 and G34 is indicated by dashed arrows. Functionally important loop nucleotides are indicated by bolded labels and rectangles.