Conformational dynamics of the human propeller telomeric DNA quadruplex on a microsecond time scale

Abstract

The human telomeric DNA sequence with four repeats can fold into a parallel-stranded propeller-type topology. NMR structures solved under molecular crowding experiments correlate with the crystal structures found with crystal-packing interactions that are effectively equivalent to molecular crowding. This topology has been used for rationalization of ligand design and occurs experimentally in a number of complexes with a diversity of ligands, at least in the crystalline state. Although G-quartet stems have been well characterized, the interactions of the TTA loop with the G-quartets are much less defined. To better understand the conformational variability and structural dynamics of the propeller-type topology, we performed molecular dynamics simulations in explicit solvent up to 1.5 μs. The analysis provides a detailed atomistic account of the dynamic nature of the TTA loops highlighting their interactions with the G-quartets including formation of an A:A base pair, triad, pentad and hexad. The results present a threshold in quadruplex simulations, with regards to understanding the flexible nature of the sugar-phosphate backbone in formation of unusual architecture within the topology. Furthermore, this study stresses the importance of simulation time in sampling conformational space for this topology.

Figure 1.

Conformational dynamics of human parallel stranded propeller-type quadruplex DNA on different timescale assessed by pairwise RMSD matrices. The minimum and maximum was assigned to a linear colour bar to indicate the variation in RMSD. Owing to large sample size, the trajectory for (a) 0–10 ns was sampled at δt = 1 ps; (b) 0–100 ns was sampled at δt = 10 ps; (c) 0–1000 ns was sampled at δt = 100 ps; (d) 0–1500 ns was sampled at δt = 150 ps. The matrix of 10 ns trajectory shows relaxation of coordinates after first ∼2 ns. The high RMSD (yellow stripes) in 100 ns matrix showed that the trajectory in this duration was still in equilibration. It is evident from matrix (c and d) that the trajectory equilibrates at ∼300 ns. A large conformational transition observed at ∼720 ns is owing to the formation of a transient triad.

Figure 2.

Thermal mobility in the parallel stranded propeller-type quadruplex. A comparison of atomic fluctuations in the (a) crystal structure and (b) MD simulation is represented as wireframe. The mobility distribution was calculated using B-factors from the crystal structure (PDB id 1KF1) and averaged RMSF from the MD simulation. The stable central G-quartets (blue) are surrounded by more mobile loops (red). This is also illustrated in (c). The mobility of the loops is independent of each other. (d) Loop1 (red) and Loop2 (green) exhibit higher mobility than the Loop3 (blue).

Figure 3.

Conformational variability in the backbone of the parallel stranded propeller-type quadruplex. The arrows indicate similar values for different bases.

Figure 4.

Time development of A:A base pair and A:A:A triad on the terminal quadruplex. Scattergram of glycosidic chi angle versus sugar pucker angle of (a) Ade1, (b) Ade7 and (c) Ade13. (d) The syn conformation of Ade1 and anti conformation of Ade13 facilitates reverse Watson and Crick A:A base pairing on terminal quartet. (e) Ade7 flips to syn conformation and interacts with Ade1 to form A1:A7:A13 triad. The K⁺ ion is represented as purple sphere.

Figure 5.

Representation of pentad and hexad alignment. The minimum distance plot of (a) Thy5 and Gua3 and (b) Thy17 and Gua15 from the middle quartet. A pentad is formed when either Thy5 or Thy17 is in plane with the quartet and within hydrogen bonding distance. A hexad is formed when both Thy5 and Thy17 simultaneously align with the middle quartet and form hydrogen bonds. Structural representation of (c) Thy17 interaction through its Watson–Crick face via sheared hydrogen bonds with Gua15 to form pentad with middle quartet. (d) Concurrent alignment of Thy5 and Thy17 with middle quartet through Watson–Crick face forms a T:(GGGG):T hexad. The first 300 ns are highlighted as a yellow box, and an arbitrary line at 3.5 Å is drawn to highlight hydrogen bond formation. The K⁺ ion is represented as purple sphere.

Figure 6.

Role of bulk water and ions in stabilization of the topology. Additional K⁺ ions positioned (a) between the terminal quartet and the A:A diad and (b) on top of the A:A diad. This is analogous to cation bonding between the quartets. (c) K⁺ ion coordination with the backbone atoms and water molecules. Ion capture in the loops occurs when an electronegative sink is generated owing to the close proximity of phosphate backbone atoms. The ion helps in stabilizing the inter-phosphate repulsion. (d) Spine of hydration as observed in a snapshot during MD simulation. The hydration networks are similar to those observed in quadruplex crystal structures. The K⁺ ion is represented as purple sphere, whereas the solvent (water) is coloured red.

Figure 7.

Ensemble of conformations identified via clustering analysis. RMSD-based clustering, with a cut-off of 3.0 Å, over equilibriated trajectory identified six clusters. Cartoon representations of top and side view of cluster centres are illustrated. The orange and blue clusters are transitional substates observed within the black cluster, whereas the red cluster appears within the pink cluster.

Figure 8.

Comparison of the loops in the (a) simulated structure (pink) with the X-ray (blue) and the NMR structure (green). The superimposition highlights the similarity between the structures from (b) loop1 and (c) Loop3. The snapshot from the simulation was extracted at 1481 ns.