Structures of 1:1 and 2:1 complexes of BMVC and MYC promoter G-quadruplex reveal a mechanism of ligand conformation adjustment for G4-recognition

Abstract

BMVC is the first fluorescent probe designed to detect G-quadruplexes (G4s) in vivo. The MYC oncogene promoter forms a G4 (MycG4) which acts as a transcription silencer. Here, we report the high-affinity and specific binding of BMVC to MycG4 with unusual slow-exchange rates on the NMR timescale. We also show that BMVC represses MYC in cancer cells. We determined the solution structures of the 1:1 and 2:1 BMVC-MycG4 complexes. BMVC first binds the 5'-end of MycG4 to form a 1:1 complex with a well-defined structure. At higher ratio, BMVC also binds the 3'-end to form a second complex. In both complexes, the crescent-shaped BMVC recruits a flanking DNA residue to form a BMVC-base plane stacking over the external G-tetrad. Remarkably, BMVC adjusts its conformation to a contracted form to match the G-tetrad for an optimal stacking interaction. This is the first structural example showing the importance of ligand conformational adjustment in G4 recognition. BMVC binds the more accessible 5'-end with higher affinity, whereas sequence specificity is present at the weaker-binding 3'-site. Our structures provide insights into specific recognition of MycG4 by BMVC and useful information for design of G4-targeted anticancer drugs and fluorescent probes.

Figure 1.

(A) MycG4, the major G-quadruplex formed in the MYC promoter NHE III₁ in K⁺ solution, a parallel-stranded structure with a 1:2:1 loop-length arrangement. Red box = guanine, green ball = adenine, blue ball = thymine. (B) Structure of BMVC molecule with numbering.

Figure 2.

Imino proton region of the 1D ¹H NMR titration spectrum of MycG4 with BMVC in pH 7, 95 mM K⁺ solution, at 25 °C. The assignments of imino protons from free MycG4 DNA are colored in black, 1:1 BMVC–MycG4 complex in red, 2:1 BMVC–MycG4 complex in blue. The H1 protons of BMVC molecules (BH1 in the 5'-end complex or BH1* in the 3'-end complex) are also shown in the spectra. The BMVC/MycG4 ratios are shown on the left side of spectra.

Figure 3.

(A) The expanded H1-H1 region of the 2D-NOESY spectrum at BMVC/MycG4 ratio of 0.5. Exchange cross-peaks of imino protons between free DNA and 1:1 BMVC–MycG4 complex are labeled with the corresponding residue numbers. (B) The expanded H8/H6-H1′ region of the 2D-NOESY spectrum of the 1:1 complex at BMVC/MycG4 ratio of 0.5. Complete sequential assignment pathway is shown. The missing connectivity is labeled with red asterisk. (C) The expanded 2D-NOESY spectra of the 1:1 complex, showing some examples of the intramolecular BMVC NOEs (left) and intermolecular NOEs between BMVC with the 5′-end of MycG4 (right). Conditions: pH 7, 95 mM K⁺, 25 °C, mixing time 200 ms.

Figure 4.

(A) Δppm 1, the MycG4 proton chemical shift difference between the free MycG4 and 1:1 BMVC–MycG4 complex and (D) Δppm 2, the MycG4 proton chemical shift difference between the 1:1 and 2:1 BMVC−MycG4 complexes. The residue numbers of MycG4 are shown. Schematic diagrams of the inter- and intra-molecular NOEs of the 5'-end complex in the 1:1 complex (B) and of the 3′-end complex in the 2:1 complex (C). Thin, medium, and thick lines correspond to strong, medium and weak NOE interactions, respectively.

Figure 5.

(A-B) A representative model of the NMR-refined 1:1 BMVC–MycG4 complex structure (the 5'-end complex) shown in two different views. (PDB ID 6JJ0) (C) top and (D) side view of the BMVC-induced binding pocket at the 5′-end of MycG4. BMVC molecules are shown in yellow. Guanines are in green, adenines in magenta and thymines in cyan.

Figure 6.

(A) The expanded H1-H1 region of the 2D-NOESY spectrum at BMVC/MycG4 ratio 1.5. Exchange cross-peaks of imino protons between the 1:1 BMVC–MycG4 complex and 2:1 BMVC–MycG4 complex are labeled with the corresponding residue numbers. Condition: pH 7, 95 mM K⁺, 25 °C. (B) The expanded H8/H6-H1′ region of the 2D-NOESY spectrum of the 2:1 BMVC–MycG4 complex at BMVC/MycG4 ratio of 1.5:1. The complete sequential assignment pathway is shown. The missing connectivity is labeled with red asterisks. The assignment of the 2:1 BMVC–MycG4 complex is labeled with superscript b. Condition: pH 7, 95 mM K⁺, 5 °C. (C) The expanded 2D-NOESY spectra of the 2:1 BMVC–MycG4 complex at BMVC/MycG4 ratio of 1.5:1, showing the intermolecular NOEs between BMVC and the 3′-end of MycG4. Condition: pH 7, 95 mM K⁺, 5 °C, mixing time 200 ms.

Figure 7.

(A, B) A representative model of the NMR-refined 2:1 BMVC–MycG4 complex structure shown in two different views. (PDB ID 6O2L) (C) Top and (D) side views of the BMVC-induced binding pocket at the 3′-end of MycG4. BMVC molecules are shown in yellow. Guanines are in green, adenines in magenta, and thymines in cyan.

Figure 8.

The stacking view of the 5′-end MycG4 G-tetrad with the contracted-form (A) or extended-form BMVC (B). BMVC is shown in magenta. (C) Proton distances between 5′-end G-tetrad with contracted-form or extended-form BMVC.

Figure 9.

Imino proton regions of the 1D ¹H NMR titration spectra of BMVC with various 5′-end modified (A) or 3′-end modified (B) MycG4 sequences in pH 7, 95 mM K⁺ solution, at 25 °C. The modified sequences are shown above the spectra with modified residues colored in green. Imino protons arising from the 1:1 or 2:1 complex formation are marked with asterisks in black or red, respectively.

Figure 10.

BMVC represses MYC expression in MCF-7 breast cancer cells. (A) BMVC lowers Myc protein levels but not glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels as shown by western blots. 0.1% DMSO is used as control. Cells treated with DMSO for 72 hr are shown in the left 2 lanes (replicates). Two concentrations of BMVC are added to different cell samples with difference treatment times (right 8 lanes). (B) qRT-PCR results showing BMVC decreases MYC mRNA levels. The BMVC-exposure time is shown to the right of the figure. (n = 3 biologically independent samples. Error bars represent mean ± s.d. *P < 0.05, **P < 0.01 by Student's unpaired t-test).