Polarizable Molecular Dynamics Simulations of Two c-kit Oncogene Promoter G-Quadruplexes: Effect of Primary and Secondary Structure on Loop and Ion Sampling

Abstract

G-quadruplexes (GQs) are highly ordered nucleic acid structures that play fundamental roles in regulating gene expression and maintaining genomic stability. GQs are topologically diverse and enriched in promoter sequences of growth regulatory genes and proto-oncogenes, suggesting that they may serve as attractive targets for drug design at the level of transcription rather than inhibiting the activity of the protein products of these genes. The c-kit promoter contains three adjacent GQ-forming sequences that have proposed antagonistic effects on gene expression and thus are promising drug targets for diseases such as gastrointestinal stromal tumors, mast cell disease, and leukemia. Because GQ stability is influenced by primary structure, secondary structure, and ion interactions, a greater understanding of GQ structure, dynamics, and ion binding properties is needed to develop novel, GQ-targeting therapeutics. Here, we performed molecular dynamics simulations to systematically study the c-kit2 and c-kit* GQs, evaluating nonpolarizable and polarizable force fields (FFs) and examining the effects of base substitutions and cation type (K⁺, Na⁺, and Li⁺) on the dynamics of their isolated and linked structures. We found that the Drude polarizable FF outperformed the additive CHARMM36 FF in two- and three-tetrad GQs and solutions of KCl, NaCl, and LiCl. Drude simulations with different cations agreed with the known GQ stabilization preference (K⁺ > Na⁺ > Li⁺) and illustrated that tetrad core-ion coordination differs as a function of cation type. Finally, we showed that differences in primary and secondary structure influence loop sampling, ion binding, and core-ion energetics of GQs.

Figure 1.

G-rich region of the c-kit promoter region. (A) Primary sequence of the G-rich region, including three GQ-forming sequences (c-kit2: red, c-kit*: purple, and c-kit1: blue). Bolded portions of the primary sequence indicate the sequences used to construct the linked c-kit2/c-kit* complex. (B) Energy-minimized c-kit2/c-kit* structure, rendered in cartoon. Core guanines are rendered as sticks. (C) Cartoon representation of experimental c-kit2, c-kit*, and c-kit1 GQ structures with bound K⁺ ions (gold). The guanine bases of the GQ core are colored by tetrad (1 – red, 2 – blue, and 3 – green).

Figure 2.

Structure and sequence of the c-kit2 and c-kit* GQs. (A) Cartoon representation of starting c-kit2 structures, highlighting bound K⁺ ions (colored gold) and nucleotides of the long propeller loop (residues 9–13; colored light blue). Residues 10, 12, and 21 vary in the c-kit2 structures and are represented as colored sticks and orange text. (B) Cartoon representation of starting c-kit* structure, highlighting a bound K⁺ ion (colored gold) and structurally important linker nucleotides (Gua10 and Cyt18) in colored stick representations and orange text. The guanine bases of the GQ cores are colored by tetrad: 1 – red, 2 – blue, and 3 – green (in c-kit2 GQs). O6 atoms pointing inward to coordinate K⁺ are colored orange.

Figure 3.

Drude K⁺ interaction maps for c-kit2 GQs. Ion sampling around c-kit2 GQs is shown at an occupancy threshold of ≥1% and the displayed percentages indicate the persistence of each ion at that location throughout the three replicate simulations.

Figure 4.

Ion-tetrad relative distance distributions and core-ion clusters show the position of the bound ions in the tetrad core. All distributions are centered on the tetrad core center of mass (relative distance = 0 Å). Ion-tetrad relative distances for all replicates were combined to produce distributions for KCl, NaCl, and LiCl. Cartoon renderings of the top tetrad core-ion cluster in each ion type (right) to illustrate common local alignment in these systems.

Figure 5.

Ion interaction maps around the T12/T21 c-kit2 GQ with the Drude-2017 FF from simulations of the isolated GQs in KCl, NaCl, and LiCl. The isosurface value for ion sampling was set at an occupancy threshold of ≥1%. The displayed percentages reflect the persistence of each ion at the indicated location across the three replicate simulations, expressed as the fraction of snapshots in which an ion was aligned with the tetrad stem (see Methods).

Figure 6.

Central structures of each of the five clusters produced by RMSD-based clustering of the c-kit* 3’-tail nucleotides (Cyt18-Cyt19-Gua20-Gua21-Cyt22). Clustering was performed on the pooled Drude simulation trajectories, thus reflecting the entire simulation ensemble. The starting GQ structure (grey) is overlaid with the central structure from each cluster. For reference, the GQ backbone is rendered as a cartoon tube and loop nucleotides are shown in sticks. Percentages denote the occupancy of each cluster.

Figure 7.

Structural characterization of the c-kit2 WT GQ in the linked c-kit2/c-kit* system. (A) Per-nucleotide RMSD, (B) per-nucleotide RMSF, and (C) central structures of the top three clusters of the c-kit2 GQ from RMSD-based clustering with associated occupancy percentages. Error bars in panels (A) and (B) represent the standard deviation of the averages of three replicate simulations.

Figure 8.

Structural characterization of the c-kit* GQ in the linked c-kit2/c-kit* system. (A) Per-nucleotide RMSD, (B) per-nucleotide RMSF, and (C) central structures of the top three clusters of the c-kit* GQ from RMSD-based clustering with associated occupancy percentages. Error bars in panels (A) and (B) represent the standard deviation of the averages of three replicate simulations.

Figure 9.

Ion interaction maps in the linked c-kit2/c-kit* GQ system. Ion sampling around the (A) c-kit2 and (B) c-kit* GQs are shown with an occupancy threshold of ≥1%.