Not so crystal clear: the structure of the human telomere G-quadruplex in solution differs from that present in a crystal

Abstract

The structure of human telomere DNA is of intense interest because of its role in the biology of both cancer and aging. The sequence [5'-AGGG(TTAGGG)3] has been used as a model for telomere DNA in both NMR and X-ray crystallographic studies, the results of which show dramatically different structures. In Na+ solution, NMR revealed an antiparallel G-quadruplex structure that featured both diagonal and lateral TTA loops. Crystallographic studies in the presence of K+ revealed a flattened, propeller-shaped structure featuring a parallel-stranded G-quadruplex with symmetrical external TTA loops. We report the results of biophysical experiments in solution and computational studies that are inconsistent with the reported crystal structure, indicating that a different structure exists in K+ solutions. Sedimentation coefficients were determined experimentally in both Na+ and K+ solutions and were compared with values calculated using bead models for the reported NMR and crystal structures. Although the solution NMR structure accurately predicted the observed S-value in Na+ solution, the crystal structure predicted an S-value that differed dramatically from that experimentally observed in K+ solution. The environments of loop adenines were probed by quantitative fluorescence studies using strategic and systematic single-substitutions of 2-aminopurine for adenine bases. Both fluorescence intensity and quenching experiments in K+ yielded results at odds with quantitative predictions from the reported crystal structure. Circular dichroism and fluorescence quenching studies in the presence of the crowding agent polyethylene glycol showed dramatic changes in the quadruplex structure in K+ solutions, but not in Na+ solutions, suggesting that the crystal environment may have selected for a particular conformational form. Molecular dynamics simulations were performed to yield model structures for the K+ quadruplex form that are consistent with our biophysical results and with previously reported chemical modification studies. These models suggest that the biologically relevant structure of the human telomere quadruplex in K+ solution is not the one determined in the published crystalline state.

Figure 1

Sequences and structures of human telomere DNA. The sequences used in this study are shown at the top. The unmodified 22 nt sequence is listed first. Singly substituted oligonucleotides containing 2-aminopurine (‘Ap’) are shown with the position of the substitution in bold. (a) NMR-derived structure of the human telomere DNA sequence in Na⁺ reported by Wang and Patel (22). (b) Structure of human telomere DNA in Na⁺ derived from crystallographic studies reported by Parkinson et al. (23).

Figure 2

Results from sedimentation velocity experiments and molecular dynamic simulations. Sedimentation coefficient distributions as determined by the program Sedfit (43) are shown for Na⁺ solutions (a) or K⁺ solutions (b), yielding the values shown in Table 1. Data for three different loading concentrations are shown (5.1, 2.55 and 1.275 μM strand). The narrower distributions in red were calculated from molecular dynamics trajectories for simulations based on the Wang–Patel (22) structure (a) or the Parkinson–Lee–Neidle structure (23) (b).

Figure 3

Results of fluorescence studies. The results of fluorescence intensity measurements (a and c) or fluorescence quenching studies using acrylamide (b and d) are shown for oligonucleotides containing 2-aminopurine. The top row (a and b) shows results obtained in Na⁺, while the bottom row (c and d) shows results obtained in K⁺. The colors indicate the position of the 2-aminopurine substitution: Ap1, black; Ap7, red; Ap13, green; Ap19, blue.

Figure 4

Effect of the crowding agent polyethyleneglycol (PEG) on quadruplex structure. The top row (a and b) shows results obtained in Na⁺. The bottom row shows results obtained in K⁺. CD spectra are shown in (a and c), while acrylamide fluorescence quenching curves are shown in (b and d). CD spectra were recorded with no PEG (black curves) to a maximum of 1.4 M PEG (cyan curves). Fluorescence quenching experiments were performed in 1.4 M PEG.

Figure 5

Alternative structures for the human telomere quadruplex in K⁺ solutions. Top panel: minimized structures (A–G) built with the properties listed in Table 2. Bottom panel: schematics of the quadruplex structures shown in the top panel as viewed from above are shown. Green circles represent 5′ to 3′ strand direction toward the reader and red circles indicate 5′ to 3′ strand direction pointing away from the reader. The lines representing the loops are topologically correct.