Structures of mithramycin analogues bound to DNA and implications for targeting transcription factor FLI1

Abstract

Transcription factors have been considered undruggable, but this paradigm has been recently challenged. DNA binding natural product mithramycin (MTM) is a potent antagonist of oncogenic transcription factor EWS-FLI1. Structural details of MTM recognition of DNA, including the FLI1 binding sequence GGA(A/T), are needed to understand how MTM interferes with EWS-FLI1. We report a crystal structure of an MTM analogue MTM SA-Trp bound to a DNA oligomer containing a site GGCC, and two structures of a novel analogue MTM SA-Phe in complex with DNA. MTM SA-Phe is bound to sites AGGG and GGGT on one DNA, and to AGGG and GGGA(T) (a FLI1 binding site) on the other, revealing how MTM recognizes different DNA sequences. Unexpectedly, at sub-micromolar concentrations MTMs stabilize FLI1-DNA complex on GGAA repeats, which are critical for the oncogenic function of EWS-FLI1. We also directly demonstrate by nuclear magnetic resonance formation of a ternary FLI1-DNA-MTM complex on a single GGAA FLI1/MTM binding site. These biochemical and structural data and a new FLI1-DNA structure suggest that MTM binds the minor groove and perturbs FLI1 bound nearby in the major groove. This ternary complex model may lead to development of novel MTM analogues that selectively target EWS-FLI1 or other oncogenic transcription factors, as anti-cancer therapeutics.

Figure 1.

Chemical structures of mithramycin, its analogues and chromomycin A3.

Figure 2.

Structure of MTM SA–Trp–DNA complex and the ligand–DNA interactions. (A) Crystal structure of MTM SA–Trp–DNA complex. The two molecules of MTM analogue are shown as light-gray and dark-gray sticks, the DNA is in orange, the Zn²⁺ ion and its coordinating waters are shown as red and yellow balls, respectively. (B) The zoomed-in view of the Zn²⁺ coordination and the GC motif recognition. (C) A schematic of the interactions in the MTM SA–Trp–DNA complex.

Figure 3.

Crystal structures of MTM SA–Phe–DNA complexes. (A) The MTM SA–Phe bound to the first GG register of the AGGGTACCCT oligomer. (B) The MTM SA–Phe bound to the second GG register of the AGGGTACCCT oligomer in the same complex as in panel A. (C) The MTM SA–Phe bound to the first GG register of the AGGGATCCCT oligomer. (D) The MTM SA–Phe bound to the second GG register of the AGGGATCCCT oligomer in the same complex as in panel C.

Figure 4.

A schematic of the interactions in the complex of MTM SA–Phe with the first GG register of AGGGTACCCT DNA.

Figure 5.

A schematic of the interactions in the complex of MTM SA–Phe with the second GG register of AGGGTACCCT DNA.

Figure 6.

Formation of a ternary FLI1 DBD–DNA–MTM complex. (A) The stabilizing effect of MTM and its analogues at 1 μM and lower concentrations on the FLI DBD complexes with AA(GGAA)₆ DNA upon heparin challenge. (B) The HSQC NMR spectra of FLI1–DBD–DNA complex in the absence (red) and in the presence (blue) of MTM SA–Trp. (C) The HSQC NMR spectra of FLI1–DBD in the absence (red) and in the presence (blue) of MTM SA–Trp.

Figure 7.

The crystal structure of FLI1 DBD in complex with the 11-mer DNA GACCGGAAGTG.

Figure 8.

DNA structural parameters for MTM SA–Phe–DNA and FLI1 DBD–DNA complexes. The sites of the bound MTM SA–Phe and FLI1 DBD are marked by gray arrows. Panels A and B display buckle and propeller (A) and major and minor groove widths (B) for the complex of MTM SA–Phe with AGGGTACCCT. Panels C and D display buckle and propeller (C) and major and minor groove widths (D) for the complex of MTM SA–Phe with AGGGATCCCT. Panels E and F display buckle and propeller (E) and major and minor groove widths (F) for the complex of FLI1 DBD with GACCGGAAGTG.

Figure 9.

Superimposition of MTM analogues in different complexes with DNA. (A) Superimposition of MTM SA–Trp (light-gray)-DNA and MTM SA–Phe (dark-gray)-AGGGTACCCT (first GG register). (B) Superimposition of MTM SA–Phe (dark-gray)-AGGGTACCCT (first GG register) with MTM SA–Phe (light-gray)-AGGGTACCCT (second GG register).

Figure 10.

Structural models of the MTM SA–Phe–DNA–FLI1 DBD complex with GGAA repeats. (A) The MTM analogue bound to the upstream GG site. (B) The MTM analogue bound to the same GG site as the FLI1 DBD. (C) The MTM analogue bound to the downstream GG site. The respective binding sites for the MTM analogue and the FLI1 DBD are shown schematically on the bottom.