Na(+) and K(+) allosterically regulate cooperative DNA binding by the human progesterone receptor

Abstract

Cooperativity is a common mechanism used by transcription factors to generate highly responsive yet stable gene regulation. For the two isoforms of human progesterone receptor (PR-A and PR-B), differences in cooperative DNA binding energetics may account for their differing transcriptional activation properties. Here we report on the molecular origins responsible for cooperativity, finding that it can be activated or repressed with Na(+) and K(+), respectively. We demonstrate that PR self-association and DNA-dependent cooperativity are linked to a monovalent cation binding event and that this binding is coupled to modulation of receptor structure. K(+) and Na(+) are therefore allosteric effectors of PR function. Noting that the apparent binding affinities of Na(+) and K(+) are comparable to their intracellular concentrations and that PR isoforms directly regulate the genes of a number of ion pumps and channels, these results suggest that Na(+) and K(+) may additionally function as physiological regulators of PR action.

Figure 1. Schematic of PR isoform primary sequence

Functional regions are as indicated: HBD, hormone binding domain; DBD, DNA binding domain; H, hinge; AF, activation functions; BUS, B-unique sequence. PR-A is defined as amino acids 165-933.

Figure 2. Schematic of selected assembly states for PR:PRE₂ interactions

Dimer binding pathway: Circles represent hormone-bound PR monomers. Squares represent PR solution dimers or PR bound to the PRE₂ promoter (k). Binding at multiple response elements is accompanied by an inter-site cooperative interaction (k_c). Events potentially associated with cooperativity are indicated by protein-protein contacts and bending of promoter DNA. Arrow refers to the direction of transcriptional start site.

Figure 3. Sedimentation velocity analysis of PR-B in either NaCl or KCl

Initial loading concentration of PR-B for both NaCl (filled squares) and KCl (line) was 1.0 μM. G(s*) distributions were determined by analysis of successive scans taken in each salt. Vertical lines indicate the weight-average sedimentation coefficient determined by analysis of each g(s*) distribution in NaCl (dotted line) and KCl (solid line). In addition to either 50 mM NaCl or KCl, buffer conditions were 20 mM Tris, pH 8.0, 2.5 mM MgCl₂, 1 mM CaCl₂, 1 mM DTT, and 10⁻⁵ M progesterone at 4°C.

Figure 4. PR-B sedimentation equilibrium in 50 mM KCl

Panels A-C represent each initial loading concentration: 0.9 μM (Panel A), 0.45 μM (Panel B), and 0.25 μM (Panel C). Symbols represent PR-B absorbance at 14,000 rpm (filled squares), 18,000 rpm (open circles), and 21000 rpm (filled triangles). Solid lines represent the best fit model (monomer-dimer-pentamer) from simultaneous analysis of all nine data sets. Square root of the variance was 0.004 absorbance units. Panels D-F show residuals from the monomer-dimer-pentamer equilibrium model plotted as a change in absorbance versus radius²/2. For the sake of clarity only every other residual point is plotted. Buffer conditions were 20 mM Tris, pH 8.0, 50 mM KCl, 2.5 mM MgCl₂, 1 mM CaCl₂, 1 mM DTT, and 10⁻⁵ M progesterone at 4°C.

Figure 5. Calculated probabilities for PR-B solution assembly states in 50 mM NaCl or KCl

Shown are the probabilities for PR-B monomers (dotted line), dimers (solid line) and pentamers (dashed line) in solutions containing either 50 mM NaCl (blue) or 50 mM KCl (red).

Figure 6. Representative quantitative footprint titration of PR-B binding to the PRE₂ promoter in KCl and individual site binding curves obtained for PR-B binding to both the PRE₂ and PRE₁₋ promoters in KCl and NaCl

Panel A – Representative footprint image of PR-B binding to the PRE₂ promoter in 50 mM KCl. Position of the two PREs (site 1 – filled rectangle; site 2 – open rectangle) are indicated in the schematic to the right of the image. Panel B – All individual site binding isotherms for PR-B binding to site 1 (filled cirles) and 2 (open circles) of the PRE₂ promoter and site 1 (open rectangle) of the PRE₁₋ promoter in 50 mM KCl. Also shown are the best fit lines from the model presented in Figure 2 describing binding to the PRE₂ (solid line) and PRE₁₋ (dotted line) promoters. The standard deviation of the fit was 0.074 apparent fractional saturation units. Panel C – All individual site binding isotherms from matched experiments for PR-B binding to the PRE₂ and PRE₁₋ promoters in 50 mM NaCl. Symbols and lines are as indicated in Panel B. The standard deviation of the fit was 0.080 apparent fractional saturation units.

Figure 7. Measured PR-B weight average s plot as a function of ion type and ion concentration

Initial PR-B loading concentration was 1.0 μM. The weight average sedimentation coefficient was determined as implemented in the program DCDT+. Error estimates represent one standard deviation as reported by DCDT+. Solution conditions minus MCl were 20 mM Tris, pH 8.0, 2.5 mM MgCl₂, 1 mM CaCl₂, 1 mM DTT, 10⁻⁵ M progesterone at 4°C. The concentration of MCl was either 10 mM (squares, solid line), 50 mM (open circles, dashed line) or 300 mM (triangles, dotted line). M⁺ is as indicated on the plot.

Figure 8. Measured PR-A weight average s plot as a function of ion type and ion concentration

Initial PR-A loading concentration was 1.0 μM. The weight average sedimentation coefficient was determined as implemented in the program DCDT+. Error estimates represent one standard deviation as reported by DCDT+. Solution conditions minus MCl were 20 mM Tris, pH 8.0, 2.5 mM MgCl₂, 1 mM CaCl₂, 1 mM DTT, 10⁻⁵ M progesterone at 4°C. The concentration of MCl was either 100 mM (squares, solid line), 200 mM (open circles, dashed line) or 300 mM (triangles, dotted line). M⁺ is as indicated on the plot.

Figure 9. Time course for trypsin digestion of PR-B in NaCl vs KCl and mapped proteolytic fragments

1 μM PR-B was digested using trypsin at 500:1 protein to enzyme mass ratio. Reactions were carried out at 4°C in 20 mM Tris, pH 8.0, 2.5 mM MgCl₂, 1 mM CaCl₂, 1 mM DTT, 10⁻⁵ M progesterone and 300 mM MCl. Reactions were allowed to proceed for 90 minutes with samples taken as a function of time. Resultant peptides were probed using immunoblot analysis, and detected using anti-HBD antibody. (*) fragment lengths are estimated using immunoblot analysis and apparent molecular mass.