MD simulations of ligand-bound and ligand-free aptamer: molecular level insights into the binding and switching mechanism of the add A-riboswitch

Abstract

Riboswitches are structural cis-acting genetic regulatory elements in 5' UTRs of mRNAs, consisting of an aptamer domain that regulates the behavior of an expression platform in response to its recognition of, and binding to, specific ligands. While our understanding of the ligand-bound structure of the aptamer domain of the adenine riboswitches is based on crystal structure data and is well characterized, understanding of the structure and dynamics of the ligand-free aptamer is limited to indirect inferences from physicochemical probing experiments. Here we report the results of 15-nsec-long explicit-solvent molecular dynamics simulations of the add A-riboswitch crystal structure (1Y26), both in the adenine-bound (CLOSED) state and in the adenine-free (OPEN) state. Root-mean-square deviation, root-mean-square fluctuation, dynamic cross-correlation, and backbone torsion angle analyses are carried out on the two trajectories. These, along with solvent accessible surface area analysis of the two average structures, are benchmarked against available experimental data and are shown to constitute the basis for obtaining reliable insights into the molecular level details of the binding and switching mechanism. Our analysis reveals the interaction network responsible for, and conformational changes associated with, the communication between the binding pocket and the expression platform. It further highlights the significance of a, hitherto unreported, noncanonical W:H trans base pairing between A73 and A24, in the OPEN state, and also helps us to propose a possibly crucial role of U51 in the context of ligand binding and ligand discrimination.

FIGURE 1.

Schematic representation of the (A) crystal structure of aptamer domain of adenine riboswitch, with ligand-binding domain; (B) ligand-binding domain of average OPEN state structure and CLOSED state structure.

FIGURE 2.

RMSD plots for (A) trajectory of OPEN and CLOSED state with respect to their initial structure; (B) trajectory of OPEN and CLOSED state for the dome region (stretches: 25–45 and 56–70) shown in solid line, and for the rest of the aptamer domain is shown in dashed line; (C) trajectory for OPEN state with respect to the average CLOSED state structure (full aptamer, dome region, and rest of the region shown in different colors); and (D) RMSF for the C1′ atoms of the nucleotides averaged over the time course of simulation. Structural regions are indicated by the color bar as per the scheme used in Figure 1A.

FIGURE 3.

Dial plots showing variation of torsion angles χ, ζ, and δ during the simulations. In the circular plots, the radial axis signifies time, with the origin as 0 psec of the production run and progressing outward, and the angular axis represents the value of torsion angles with the range spanning as 0 to 2π in anticlockwise direction with 0 being at the right side of the plot. The histograms plotted at the periphery of the circular plots denote the frequency of the values spanned by torsion angles.

FIGURE 4.

Dynamic cross-correlation maps calculated as time averaged for the centroid of base pairs over the simulation. Regions 1, 2, and 3 indicate the expected positive correlation between nucleotide pairs constituting the helices P1, P2, and P3, respectively. Regions 4, 5, and 6 indicate intrajunction self-correlations of J1-2, J2-3, and J3-1, respectively. Regions 7, 8, and 9 reveal the nature of inter junction correlations, namely, J1-2/J2-3, J2-3/J3-1, and J3-1/J1-2, respectively. Since the junction nucleotides are expected to move along with the neighboring nucleotides in their adjoining helices, the regions are marked in the figure by appropriately including portions of the latter. The correlation pattern in the intraloop nucleotide dynamics are indicated in the regions 10 (L2) and 11 (L3), while the L2–L3 interloop correlations are shown as region 12. Region 13 shows the correlation of P1 nucleotides with the rest of the aptamer fold. The color bar along the axes indicates the structural elements to which residues belong, with color coding as in Figure 1A.

FIGURE 5.

Comparison of specific significant interactions in average OPEN and CLOSED state structures. Note change in base-pairing geometry and interactions of C53 and A55, in contrast with retention of the same for A52 and C54. Hydrogen-bond distances are given in angstroms.

FIGURE 6.

(A) Superposition of the average structures of OPEN (in blue color) and CLOSED (in red color) states with that of the crystal structure (in green color). (B) Superposition of average OPEN (in blue) and CLOSED (in red) state structures. (C) Lengthening of the P1 helix in the average structure of the OPEN state compared with that of the CLOSED state. (D) Superposed QM optimized geometries of triplets U74–Guanine–U51 (in green color) and U74–Adenine–U51 (in magenta color). C1′–C1′ distances are shown between U51 and U74. (E) Mg²⁺ ions positions captured at each 1-nsec snapshot for the OPEN and CLOSED states. (F) Tertiary contacts of the U47 shape the dynamics of the J2-3 backbone and mediate an essentially flipped out geometry of U51. (G) Anchoring of P2 and P3 to keep the roof and dome above the binding pocket in the preformed fold in the open state.

FIGURE 7.

Schematic representation of our multistate model for the binding and switching process. The preformed roof–dome architecture is not included. The nucleotides corresponding to the three junction loops, and the functionally important P1 nucleotides are shown using four different colors, respectively. The red circle represents a water molecule in a water-mediated hydrogen bond. The yellow oval represents the adenine ligand. The backbone inherits colors from the colors of the residues it connects up, while base-pairing interactions are shown in red. The black dot on the backbone indicates the phosphate group. Blue dotted lines represent weak tertiary interactions in the OPEN state, while the red dotted lines indicate tertiary interactions evolving during the process of binding. The terminal residues of the junction loops are shown having contact with the aptamer. A change in shape of the aptamers represents conformational changes.