Role of the adenine ligand on the stabilization of the secondary and tertiary interactions in the adenine riboswitch

Abstract

Riboswitches are RNA-based genetic control elements that function via a conformational transition mechanism when a specific target molecule binds to its binding pocket. To facilitate an atomic detail interpretation of experimental investigations on the role of the adenine ligand on the conformational properties and kinetics of folding of the add adenine riboswitch, we performed molecular dynamics simulations in both the presence and the absence of the ligand. In the absence of ligand, structural deviations were observed in the J23 junction and the P1 stem. Destabilization of the P1 stem in the absence of ligand involves the loss of direct stabilizing interactions with the ligand, with additional contributions from the J23 junction region. The J23 junction of the riboswitch is found to be more flexible, and the tertiary contacts among the junction regions are altered in the absence of the adenine ligand; results suggest that the adenine ligand associates and dissociates from the riboswitch in the vicinity of J23. Good agreement was obtained with the experimental data with the results indicating dynamic behavior of the adenine ligand on the nanosecond time scale to be associated with the dynamic behavior of hydrogen bonding with the riboswitch. Results also predict that direct interactions of the adenine ligand with U74 of the riboswitch are not essential for stable binding although it is crucial for its recognition. The possibility of methodological artifacts and force-field inaccuracies impacting the present observations was checked by additional molecular dynamics simulations in the presence of 2,6-diaminopurine and in the crystal environment.

Figure 1

(a) The three dimensional structure of the adenine riboswitch (PDB ID: 1Y26) generated using VMD. The bound adenine base is depicted by a CPK representation. The stems (P1, P2 & P3), the hairpin loops (L2 and L3) that cap P2 and P3 stems, and the junctions (J12, J23 & J31) that connect the three stems are color coded. (b) The secondary structure of the riboswitch, color coded similar to the three dimensional structure. The regular base pair interactions in the stems, and tertiary interactions in the loops and junctions via WC base pairing are represented by dashed lines. The hydrogen bond and the stacking interactions of the riboswitch with the adenine ligand are denoted using bold dashed and bold solid lines. For the purpose of clarity, tertiary contacts via noncanonical base pair interactions are not shown.

Figure 2

Adenine riboswitch unfolding pathway derived using single molecule force measurement studies reported by Greenleaf et al. ‘a’ is the fully folded adenine riboswitch and ‘e’ is its completely extended form. ‘b’, ‘c’ and ‘d’ are the hypothesized intermediate structures observed during the force induced unfolding. The P1 stem is disrupted as the first step to form ‘b’ followed by the loss of tertiary interactions between L2 and L3 resulting in ‘c’. After the formation of the intermediate ‘c’, the P3 stem unfolds to form ‘d’ followed by the unfolding of P2 stem leading to the unfolded structure, ‘e’. Different regions of the riboswitch are colored as in Figure 1.

Figure 3

RMS fluctuations averaged over each nucleotide computed for H1 (red line), H2 (green line), H3 (blue line), A1 (magenta line), and A2 (orange line) with the respect to the average structure along the MD trajectories. The fluctuation data (black line) calculated from the temperature factors (eq. 1) are also given for comparison. Different parts of the RNA (P1, P2, P3 and so on) are marked below the x-axis

Figure 4

Computed dynamic cross correlation map for all the pairs of the nucleotides in the riboswitch. The maps for the (a) holo forms H1 and H2 are given in the upper and lower triangular regions respectively, and (b) apo forms, A1 and A2 in the upper and lower triangular regions respectively. Highly positive (C(i,j) close to 1) are given in dark blue, no correlations (C(i,j) close to 0) are given by white and highly negative (C(i,j) close to −1) are given in dark red. Grey colored lines are drawn to differentiate the various stems, loops and the junctions. P1a and P1b represent the two strands of the stem P1 and similar nomenclature are used for stems P2 and P3.

Figure 5

Image of the hydrogen bonding (a) and stacking interactions (b) of the nucleotides of the riboswitch with the adenine ligand. (c) Probability distributions of the donor-acceptor distances of the hydrogen bonds involved in riboswitch-ligand binding. Hydrogen bond distances from the crystal structure are depicted by green lines. Images generated using VMD.

Figure 6

Autocorrelation functions of selected distances between riboswitch and adening ligand atoms in H1. Experimental autocorrelation functions are in red and fits of the decays to multiple exponential functions are in green. Included in each panel are the decays times in ns from the fits and the final squared correlation coefficient. Distances studies include (A) O2’(U22)-N7, (B) O2(U47)-N9, (C) O4(U51)-N9, (D) N3(U51)-N3, (E) N3(U74)-N1 and (F) O4(U74)-N6.

Figure 7

The snapshots of the three dimensional structures obtained from the apo simulations (A1) illustrating the opening of the J23 junction, and its movement away from the adenine regions. The initial and final (40 ns) structures are also given for comparison. The color codes used are the same as in Figure 1.