A mechanism for S-adenosyl methionine assisted formation of a riboswitch conformation: a small molecule with a strong arm

Abstract

The S-adenosylmethionine-1 (SAM-I) riboswitch mediates expression of proteins involved in sulfur metabolism via formation of alternative conformations in response to binding by SAM. Models for kinetic trapping of the RNA in the bound conformation require annealing of nonadjacent mRNA segments during a transcriptional pause. The entropic cost required to bring nonadjacent segments together should slow the folding process. To address this paradox, we performed molecular dynamics simulations on the SAM-I riboswitch aptamer domain with and without SAM, starting with the X-ray coordinates of the SAM-bound RNA. Individual trajectories are 200 ns, among the longest reported for an RNA of this size. We applied principle component analysis (PCA) to explore the global dynamics differences between these two trajectories. We observed a conformational switch between a stacked and nonstacked state of a nonadjacent dinucleotide in the presence of SAM. In the absence of SAM the coordination between a bound magnesium ion and the phosphate of A9, one of the nucleotides involved in the dinucleotide stack, is destabilized. An electrostatic potential map reveals a 'hot spot' at the Mg binding site in the presence of SAM. These results suggest that SAM binding helps to position J1/2 in a manner that is favorable for P1 helix formation.

Figure 1.

(a) Schematic of the regulatory mechanism of the SAM-I riboswitch. This model has been previously described (11–13,15). In the presence of SAM, pairing of nonadjacent regions (highlighted in blue) forms the P1 helix of the anti-anti-terminator (AAT) structure, which prevents formation of the anti-terminator (AT). Under these conditions, the ρ-independent terminator (T) hairpin can form to achieve transcriptional attenuation. (b) Three-dimensional crystallographic structure of the SAM-I riboswitch from T. tencongensis (35). A close-up is shown of the SAM binding pocket, highlighting the positions of two of the residues that are observed to contact the RNA. (c) Secondary structure representation of tertiary interactions observed in the crystallographic [Residues in red are involved in formation of the SAM binding pocket. The direction of the arrow is from 5′ to 3′. The base pairs are automatically identified using RNAVIEW (60). The structural motifs are annotated as ref. (61)].

Figure 2.

Time evolution of RMSD of the binding pocket and the ligand SAM relative to X-ray coordinates (35). In the presence of SAM (SAM_TRAJ, red), the RMSD is stable, while in the absence of SAM (WoSAM_TRAJ, blue), the RMSD increases until ∼100 ns.

Figure 3.

Clustering and principal component analysis (PCA) point toward a chopstick-like motion involving P1 and P3 helices in the absence of SAM. (a) Projections of snapshots of the WOSAM-TRAJ are plotted against the first two principal components and color coded according to a k-means clustering (k = 3): cluster 0: green; cluster 1, cyan; cluster 2, magenta.. Representative snapshots from each cluster are also shown. This plot indicates that snapshots can be broadly clustered into two groups (cluster 1 and cluster 2) with cluster 0 representing a group with characteristics similar to those of cluster 2. The projection along PC1 broadly separates the clusters, while projection along PC2 completes the separation between clusters 0 and 2. Structures of representative snapshots indicate that clusters are distinguished by a dramatic change in relative position of P1 and P3. (b) (From top to bottom) The time evolution of the first principle component of WoSAM_TRAJ. (each snapshot is color-coded as in Figure 3a). RMSD for each snapshot in WOSAM_TRAJ relative to the representative snapshots for cluster 0 (cyan curve) and for cluster 2 (magenta curve). The distance between the Center of Mass (COM) of P1 and P3 for WOSAM_TRAJ (blue) and for SAM_TRAJ (red). During the first half of WOSAM_TRAJ, P1 and P3 helices move apart (clusters 0 and 2), then they move back together during the second half of the trajectory (cluster 1).

Figure 4.

A9/U63 dinucleotide stack state monitor and the correlation with glycosidic angles for the respective residues. (a) Monitor of Lennard–Jones energy (E_VDW) of interaction between the A9 and U63 residues involved in a nonadjacent stack in SAM_TRAJ (red) and WoSAM_TRAJ (blue). Snapshots above the panel of the A9/U63 stacking geometry show that the intervals during which E_VDW is lowered correspond to the dinucleotide stacked state; (b) monitor of the glycosidic angle of residues involved in the nonadjacent stack in all the simulations. Histograms showing occupancy of the stacking states and glycosidic angles for A9 and U63 are on the right side of each figure respectively. A biphasic distribution for the A9 glycosidic angle is indicative of anharmonic motions.

Figure 5.

Relaxation of the magnesium coordination with J1/2 in the absence of SAM and its coordination with the backbone of nucleotide A9 is correlated with the A9/U63 dinucleotide stack state (Figure 4). (a) Residues near the specific magnesium binding site; (b) distance monitor between the specific binding magnesium and atoms on the phosphate backbone (Red: SAM_TRAJ, blue: WoSAM_TRAJ). Coordination of the magnesium ion with phosphates in J1/2 is stabilized in SAM_TRAJ; (c) distance monitor between the specific binding magnesium and O3′ in A9 in SAM_TRAJ. A slight shift is correlated with the non-adjacent dinucleotide stack (see text and Figure 4).

Figure 6.

Electrostatic potential maps (ESPs) highlighting two orthogonal slices through a Mg binding site. (a) ESP map for the X-ray coordinates with SAM bound. SAM is highlighted in yellow. J1/2 is highlighted in orange, J3/4 in magenta and the position of the Mg is highlighted by a green sphere. Note that phosphate groups appear as red and some electropositive functional groups as blue. A large region of negative potential appears on all sides of the Mg ion, bordered tightly by J1/2 and J3/4. (b) Close-ups of ESP in the region around the Mg site for three representative snapshots of SAM_TRAJ (left) and WOSAM_TRAJ (right). The snapshots are chosen from the centroid of each of the three main clusters. In SAM_TRAJ snapshots, the hotspot of negative potential is tightly surrounded by J1/2 and J3/4, whereas it is concentrated near J3/4 only in the absence of SAM.

Figure 7.

The distances between hydrogen bond donor and acceptor of hydrogen bonds between SAM and the SAM-I riboswitch RNA in MD trajectory (nitrogen atoms are colored in blue, and oxygen atoms are colored in red). The contacts between SAM and G11 and G58 are extremely stable during the simulation. For the hydrogen bonding monitor, see Supplementary Table 1.