Contributions of direct and indirect interactions to Ubx–Exd ternary complex formation. a) The crystal structure of the Ubx–Exd–DNA ternary complex, showing the relative binding orientation of the two proteins. The N-terminal portion of Ubx reaches across the DNA, contacting Exd and stabilizing the ternary complex. b) Schematic of wild-type and mutant Ubx proteins and the DNA sequence used for Ubx–Exd–DNA interactions. Wild-type Ubx contains the YPWM interaction peptide, while the peptide is mutated to alanines in the mutant Ubx. c–e) The ability of Ubx to recruit Exd to DNA was evaluated via EMSA (in 0.5x TBE). Both wild-type Ubx (c) and the interaction peptide mutant (d) were able to improve binding of Exd over the Ubx free condition (e). Note the lowered Exd concentration range used for wild-type Ubx. f) The dissociation constant (K_D) and the fraction occupancy at projected saturation (θ) of each condition was determined by fitting the fraction DNA bound values obtained by EMSA to the Hill binding equation. Wild-type Ubx improves Exd binding by 800-fold, 100-fold of which is due to the YPWM interaction peptide.

Ternary complex assembly by small molecules. a) Polyamides bind to the minor groove and are known to increase the width and depth of the minor groove in comparison to B-form DNA. An Im,Im,Py,Py-γ-Py,Py*,Py,Py-βala-Dp polyamide can bind site specifically to the minor groove of DNA at a physiological site. In a model of joint binding, there are no steric contacts between the minor groove bound polyamide and the major groove bound Exd (green). The four compounds used in this study are a YPWM Exd interaction peptide (1) or a nonfunctional derivative (2) conjugated to the polyamide via a propylamine linker, which was also present in the unconjugated form (3). In later experiments the linker and peptide was replaced by a methyl group (4). b) The ability of the compounds to recruit Exd to different sites was qualitatively assessed by EMSA (in 1x TBE). The effect of the compounds varied on the basis of the relative orientation and position of the polyamide and Exd binding sites (Exd binding site highlighted). The polyamide displaying the Exd interaction peptide (1) was able to recruit Exd to DNA in all binding orientations tested. The compound with no peptide (3) was still able to recruit Exd to DNA when properly oriented with respect to the Exd binding site (the Forward orientation).

Structural analysis of Hox–TALE–DNA complexes. a,b) CURVES analysis of published Hox–Exd/Pbx1–DNA complexes, compared to B-form DNA. The DNA sequence for the Ubx–Exd complex is given, with the Exd/Pbx1 binding site highlighted. The curve for Ubx–Exd does not extend to the end of the binding site due to limitations of the CURVES algorithm close to the ends of the shorter Ubx–Exd DNA. a) The major grooves of the ternary complexes (color-coded) are wider than the B-form DNA (in gray) over the center of the Exd/Pbx1 binding site, transitioning to narrower as one enters the first few nucleotides of the Hox binding site. b) The major grooves of the complexes are generally deeper than those of the B-form, likely to accommodate the DNA recognition helices of the proteins. Of note is the sudden, sharp decrease in groove depth at the central adenine of the Exd/Pbx1 binding site, indicative of a structural deformation at that location. c) Alignment of the four DNAs against B-form (gray). Only a single strand is shown for clarity. All four show a bend away from B-form structure at the central ^5′GA^3′ base step of the Exd/Pbx1 binding site.

Contribution of direct and indirect interactions to polyamide–Exd ternary complex formation. a) Exd makes a number of contacts to DNA, including many to the backbone phosphates (figure redrawn from ref 32). These contacts have the potential to be disrupted by the binding of the positively charged propylamine of compound 3 (potential range shown as purple sphere) The propylamine linker may make direct or water-mediated contacts with the DNA or protein. b) Quantitation of Exd binding to Forward DNA by optimized (0.5x TBE) EMSA. Although Exd alone shows binding at high concentrations, the presence of 1 and 4 improves the Exd–DNA interaction. The fraction of bound DNA (including the nonspecific, slower migrating species) was calculated for each Exd concentration, plotted, and fit to the Hill binding equation. The dissociation constant (K_D) and the fraction occupancy at saturation (θ) of each condition are shown. In the Forward binding orientation, addition of 4 improves the binding of Exd 10-fold. The addition of the Exd interaction peptide improves Exd recruitment a further 100-fold.