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.

Polymorphism of human telomeric quadruplex structures

“…Actually, a calculation on the hybrid-1 structure [50] gave a sedimentation coefficient (S) value of 2.09 with two K + ions bound, which is in good agreement (1.5% difference) with the experimentally observed value of 2.11 (unpublished data from Prof. John Trent at the University of Louisville). It was also shown in the same report that the addition of PEG200, which may mimic the crystal condition, can shift the conformation of wtTel22 to a parallelstranded structure [38]. A more recent study has elaborated on this observation, that the presence of a high concentration PEG200 as a crowding agent can completely shift the conformation of wtTel21 ( Figure 3A) to the parallel-stranded structure [68].…”

“…A large number of biophysical and crosslinking studies have been reported on the K + -structure of human telomeric G-quadruplexes [32][33][34][36][37][38][39][40]. Structure polymorphism of the human telomeric G-quadruplexes, particularly for the 22-nt wtTel22 sequence, has been reported by various methods.…”

“…The hybrid-type G-quadruplex structure can explain the results and predictions made in previous biophysical studies where physiologically relevant conditions were used with the wild-type human telomeric sequence. In a recent biophysical study [38] sedimentation coefficients and 2-aminopurine (2-AP) fluorescence were used to probe the Gquadruplex structure formed by wtTel22 in K + solution. The order of the accessibility of the single substitution of 2-AP as probed by fluorescence quenching was consistent with the hybrid-1 type structure.…”

“…Because the K + structure is considered to be biologically more relevant due to the higher intracellular concentration of K + , it has been the subject of intense investigation [32][33][34][35][36][37][38][39][40][41][42][43][44][45], although the Na + structure was reported more than a decade ago [46]. In 2002, a crystal structure of the parallel-stranded G-quadruplex was reported using a 22-nt human telomeric sequence in the presence of K + [47].…”

An important functional group, benzo[1,3]dioxole, of alkaloids induces the formation of the human telomeric DNA G-quadruplex

“…Melting curves of the antiparallel G-quadruplexes were obtained by recording the CD intensity at 292 nm. The heating rate was 1.0 °C min CD measurement has been extensively applied to study secondary structures of DNA [11][12][13]. The CD spectral features allow the approximate, but convenient, recognition of different DNA motifs.…”