Ion Binding Properties and Dynamics of the bcl- 2 G-Quadruplex Using a Polarizable Force Field

Abstract

G-quadruplexes (GQs) are topologically diverse, highly thermostable noncanonical nucleic acid structures that form in guanine-rich sequences in DNA and RNA. GQs are implicated in transcriptional and translational regulation and genome maintenance, and deleterious alterations to their structures contribute to diseases such as cancer. The expression of the B-cell lymphoma 2 (Bcl-2) antiapoptotic protein, for example, is under transcriptional control of a GQ in the promoter of the bcl-2 gene. Modulation of the bcl-2 GQ by small molecules is of interest for chemotherapeutic development but doing so requires knowledge of the factors driving GQ folding and stabilization. To develop a greater understanding of the electrostatic properties of the bcl-2 promoter GQ, we performed molecular dynamics simulations using the Drude-2017 polarizable force field and compared relevant outcomes to the nonpolarizable CHARMM36 force field. Our simulation outcomes highlight the importance of dipole-dipole interactions in the bcl-2 GQ, particularly during the recruitment of a bulk K⁺ ion to the solvent-exposed face of the tetrad stem. We also predict and characterize an "electronegative pocket" at the tetrad-long loop junction that induces local backbone conformational change and may induce local conformational changes at cellular concentrations of K⁺. These outcomes suggest that moieties within the bcl-2 GQ can be targeted by small molecules to modulate bcl-2 GQ stability.

Figure 1.

Structure of the bcl-2 GQ. (A) Schematic of all residues in bcl-2 GQ structure and core ions. (B) Full structure shown in cartoon with bound K⁺ ions, taken from the NMR ensemble deposited in PDB entry 2F8U. Core guanine nucleotides are colored by tetrad (Tetrad 1 – red, Tetrad 2 – blue, Tetrad 3 – green). Loop regions are colored in grey. (C) Stick representation of Gua5 and Cyt6, which stack with tetrad 1. (D) Stick representation of Ade10 and Thy15, which participate canonical base pairing to stabilize the long loop below tetrad 3.

Figure 2.

Average per-nucleotide (A) RMSD and (B) RMSF. Error bars represent the standard deviations of the averages over three replicate simulations with each FF.

Figure 3.

Central structures from the C36 simulations from RMSD-based clustering of the pooled simulation trajectories. Percentages reflect the occupancy of each cluster. Guanine tetrads are colored as in Figure 1.

Figure 4.

Central structures from the Drude-2017 simulations from RMSD-based clustering of the pooled simulation trajectories. Percentages reflect the occupancy of each cluster. Guanine tetrads are colored as in Figure 1.

Figure 5.

Average dihedral values for backbone dihedral angles of all residues. Error bars represent the standard deviation of the 10 models in the experimental NMR ensemble and the RMSF over all data points in the pooled MD trajectories using the Drude-2017 FF.

Figure 6.

Average dihedral values for glycosidic linkage (χ) and pseudorotation angles for all residues. Error bars represent the standard deviation of the 10 models in the experimental NMR ensemble and the RMSF over all data points in the pooled MD trajectories using the Drude-2017 FF.

Figure 7.

Ion interaction maps indicating the K⁺ sampling around the bcl-2 GQ, using the NMR structure for reference with the Drude FF at an occupancy threshold of ≥1%. The Gua5 residue just above the tetrad core is shown in ball-and-stick for perspective. Guanine tetrads are colored as in Figure 1. Percentages indicate the occupancy of the three K⁺ binding sites along the GQ stem axis described in the main text.

Figure 8.

The mechanism of bulk K⁺ alignment. Trajectory snapshots are shown from (A) replicate 1, (B) replicate 2, and (C) replicate 3. Time stamps are indicated above each snapshot. Guanine bases in tetrad 1 are colored in red for perspective.

Figure 9.

Changes in base dipole moments and interaction energies in response to bulk K⁺ alignment. (A) Minimum distance between any bulk K⁺ ion and Gua5 N3 (black) and Cyt6 O1P (blue), (B) dipole moment of all guanine bases in tetrad 1 and Gua5, and (C) interaction energy between tetrad bases and K⁺ bound ions shown as a running average. All data series are shown as a 100-point running average.