How mithramycin stereochemistry dictates its structure and DNA binding function

Abstract

An aureolic acid natural product mithramycin (MTM) has been known for its potent antineoplastic properties. MTM inhibits cell growth by binding in the minor groove of double-stranded DNA as a dimer, in which the two molecules of MTM are coordinated to each other through a divalent metal ion. A crystal structure of an MTM analogue, MTM SA-Phe, in the active metal ion-coordinated dimeric form demonstrates how the stereochemical features of MTM define the helicity of the dimeric scaffold for its binding to a right-handed DNA double helix. We also show crystallographically and biochemically that MTM, but not MTM SA-Phe, can be inactivated by boric acid through formation of a large macrocyclic species, in which two molecules of MTM are crosslinked to each other through 3-side chain-boron-sugar intermolecular bonds. We discuss these structural and biochemical properties in the context of MTM biosynthesis and the design of MTM analogues as anticancer therapeutics.

Fig. 1. Chemical structures of MTM and MTM SA-Phe.

Fig. 2. Structures of MTM SA-Phe alone and in complex with DNA. A. The crystal structure of an MTM SA-Phe dimer (orange and dark grey sticks) coordinated by a Mg²⁺ ion (a purple ball). Two water molecules also coordinated by the Mg²⁺ are shown as light grey balls. B. Superimposition of the structure of unbound MTM SA-Phe dimer (as in panel A; orange and dark grey sticks) with our previously reported crystal structure of MTM SA-Phe dimer bound to a DNA oligomer (PDB ID: ; 5JW0; pale yellow and green sticks). The DNA is shown in light-grey. Oxygen and nitrogen atoms of MTM SA-Phe are shown in red and blue, respectively.

Fig. 3. The crystal structure of MTM in complex with Zn²⁺ and boron. A. The structure of a (MTM)₂-Zn²⁺-B₂ complex. Two MTM portions are shown by orange and dark grey sticks, the Zn²⁺ ion is shown as a yellow ball, water as light grey balls. The boron atoms are shown in dark blue. B. A zoomed-in view of the boron bonds. C. The crystal packing interactions between two (MTM)₂-Zn²⁺-B₂ complexes. The second (MTM)₂-Zn²⁺-B₂ complex is shown by white sticks.

Fig. 4. Zoomed-in views of the divalent metal ion coordination in the structures of (MTM SA-Phe)₂-Mg²⁺ (A) and (MTM)₂-Zn²⁺-B₂ (B). The color schemes are the same as in Fig. 2 and 3.

Fig. 5. Effects of boron on DNA binding by MTM and MTM SA-Phe. A. DNA binding by MTM (5 μM dimers) in the absence of boron in Mg²⁺ (open circles), in Zn²⁺ (filled circles), in Mg²⁺ after the DNA (but not MTM) was preincubated with 20 mM boric acid for 16 hours at 21 °C (open diamonds), after MTM was preincubated with 4 mM boric acid for 4 hours (open triangles) and for 16 hours (open squares). The curves correspond to the best fits of the 1 : 1 (MTM dimer : DNA) binding isotherms with K_d < 1 μM and the fractions of MTM active in DNA binding of 1 (solid curve), 0.51 ± 0.06 (long dashes) and 0.09 ± 0.05 (short dashes) in the absence of boric acid, for the 4 hour and for the 16 hour preincubation with boric acid, respectively. B. DNA binding by MTM SA-Phe in the absence of boric acid (open circles) and after MTM SA-Phe was preincubated with 20 mM boric acid for 16 hours at 21 °C (open squares). The curve is the best fit of the 1 : 1 (MTM SA-Phe) binding isotherm, K_d = (1.6 ± 0.6) μM.