PDGFR-β Promoter Forms a Vacancy G-Quadruplex that Can Be Filled in by dGMP: Solution Structure and Molecular Recognition of Guanine Metabolites and Drugs

Abstract

Aberrant expression of PDGFR-β is associated with a number of diseases. The G-quadruplexes (G4s) formed in PDGFR-β gene promoter are transcriptional modulators and amenable to small molecule targeting. The major G4 formed in the PDGFR-β gene promoter was previously shown to have a broken G-strand. Herein, we report that the PDGFR-β gene promoter sequence forms a vacancy G-quadruplex (vG4) which can be filled in and stabilized by physiologically relevant guanine metabolites, such as dGMP, GMP, and cGMP, as well as guanine-derivative drugs. We determined the NMR structure of the dGMP-fill-in PDGFR-β vG4 in K⁺ solution. This is the first structure of a guanine-metabolite-fill-in vG4 based on a human gene promoter sequence. Our structure and systematic analysis elucidate the contributions of Hoogsten hydrogen bonds, sugar, and phosphate moieties to the specific G-vacancy fill-in. Intriguingly, an equilibrium of 3'- and 5'-end vG4s is present in the PDGFR-β promoter sequence, and dGMP favors the 5'-end fill-in. Guanine metabolites and drugs were tested and showed a conserved selectivity for the 5'-vacancy, except for cGMP. cGMP binds both the 3'- and 5'-end vG4s and forms two fill-in G4s with similar population. Significantly, guanine metabolites are involved in many physiological and pathological processes in human cells; thus, our results provide a structural basis to understand their potential regulatory functions by interaction with promoter vG4s. Moreover, the NMR structure can guide rational design of ligands that target the PDGFR-β vG4.

Figure 1.

a) The schematic of the human PDGFR-β gene promoter. The G4-forming region of NHE sequence and its modifications are shown. The guanine runs involved in the formation of the major G4 and mutations are highlighted in red and cyan, respectively. b) Schematic structure of the major PDGFR-β G4 with the sequence of Pu22m1. c) An intact G-tetrad layer including the bound guanine derivative (pink). Hoogsteen hydrogen bonds are indicated by dashed lines. d) 1D ¹H NMR titration of Pu19m2 DNA by dGMP with assignment of complex at 25 °C. e) CD spectra of Pu19m2 DNA in the presence and absence of 20 dGMP equivalents. f) A schematic model of Pu19m2 vG4s and dGMP fill-in.

Figure 2.

a) The H1′-H8 region from the 2D-NOESY spectrum of dGMP-Pu19m2 complex in H2O at 15 °C with the sequential assignment pathway, mixing time of 250 ms. Missing connectivities are labeled with asterisks. The G14H1′/A2H2 NOE cross-peak is labeled in red. b) The H8-H1 region. Intra-tetrad, inter-tetrad, and NOEs with flanking bases are labeled in red, blue, and green, respectively. The intermolecular NOE cross-peaks of bound-dGMP (dGMPa) and Pu19m2 DNA residues are marked by black boxes.

Figure 3.

Superposition of 10 lowest energy structures of the dGMP-Pu19m2 complex by NOE-restrained structure calculation: top view (left) and side view (right). Cyan indicates anti-guanine; magenta, dGMP; green, adenine; and orange, cytosine.

Figure 4.

a) Cartoon representation of the 1:1 dGMP-Pu19m2 complex (PDB ID: 6V0L). b) Side view of base stacking of dGMP to G11. A potential C1′−H1′⋯O4′ hydrogen bond is shown in dashed line. c) 5′-end and d) 3′-end views of the capping structures. Potential hydrogen-bonds are shown as dashed lines.

Figure 5.

a) and b) 1D ¹H NMR titrations of Pu19m2 DNA with various guanine derivatives. Imino protons corresponding to minor species are labeled with red asterisks. c) Chemical structures of dGMP, cGMP, and Acyclovir.