Thermodynamic analysis of progesterone receptor-promoter interactions reveals a molecular model for isoform-specific function

Abstract

Human progesterone receptors (PR) exist as two functionally distinct isoforms, PR-A and PR-B. The proteins are identical except for an additional 164 residues located at the N terminus of PR-B. To determine the mechanisms responsible for isoform-specific functional differences, we present here a thermodynamic dissection of PR-A-promoter interactions and compare the results to our previous work on PR-B. This analysis has generated a number of results inconsistent with the traditional, biochemically based model of receptor function. Specifically, statistical models invoking preformed PR-A dimers as the active binding species demonstrate that intrinsic binding energetics are over an order of magnitude greater than is apparent. High-affinity binding is opposed, however, by a large energetic penalty. The consequences of this penalty are 2-fold: Successive monomer binding to a palindromic response element is thermodynamically favored over preformed dimer binding, and DNA-induced dimerization of the monomers is largely abolished. Furthermore, PR-A binding to multiple PREs is only weakly cooperative, as judged by a 5-fold increase in overall stability. Comparison of these results to our work on PR-B demonstrates that whereas both isoforms appear to have similar DNA binding affinities, PR-B in fact has a greatly increased intrinsic binding affinity and cooperative binding ability relative to PR-A. These differences thus suggest that residues unique to PR-B allosterically regulate the energetics of cooperative promoter assembly. From a functional perspective, the differences in microscopic affinities predict receptor-promoter occupancies that accurately correlate with the transcriptional activation profiles seen for each isoform.

Fig. 1.

Schematic of selected assembly states for PR-A–PRE₂ interactions. (A) Dimer binding pathway. Circles represent hormone-bound PR-A structure, and squares represent PR-A solution dimers (k_di) or PR-A bound to the PRE₂ promoter (k₂). (B) Monomer binding pathway. Successive binding at an individual response element (k₁) is accompanied by an intrasite cooperative interaction (k_c1) represented by a transition from a filled circle to a filled square. Binding at multiple response elements is accompanied by an intersite cooperative interaction (k_c2). Arrow refers to the direction of transcriptional start site.

Fig. 2.

Quantitative footprint titration of the PRE₂ promoter and individual-site binding isotherms obtained for PR-A binding to the PRE₂ and PRE₁₋ promoters. (A) PRE₂ footprint titration. A schematic of PRE₂ promoter structure is shown at the left. Small arrows indicate the appearance of hypersensitive bands seen on both the PRE₁₋ and the PRE₂ promoters, and the large arrow indicates the appearance of bands seen only for PRE₂. (B) Symbols represent binding at site 1 of the PRE₂ promoter (red filled circles), binding at site 2 of the PRE₂ promoter (red open circles), and binding at site 2 of the PRE₁₋ promoter (blue open squares). Lines represent best fits to sites 1 and 2 of the PRE₂ (red) and site 1 of the PRE₁₋ (blue) data as resolved by a global analysis using the dimer pathway model.

Fig. 3.

Predicted distribution of each macroscopic PR-A–PRE₂ and PR-B–PRE₂ ligation state. (A) Distribution of PR-A ligation states as predicted by the dimer-binding pathway energetics (Table 1). (B) Same as A except that PR-B ligation states are displayed. Unligated PRE₂ promoter is represented by the dashed line, singly ligated promoter is represented by the solid line, doubly ligated promoter is represented by the dotted line, and the proportion of PR dimers is represented by “+.” A shaded box represents the estimated intracellular PR concentration (28).

Fig. 4.

Predicted distribution of PR–PRE₂ ligation states in the presence of equimolar concentrations of both isoforms. Simulations were carried out by using the dimer pathway energetics and assume that (i) the A- and B-isoforms heterodimerize with an average of the two homodimerization affinities, (ii) heterodimers bind a PRE with an average of the PR-A and PR-B homodimer affinity, and (iii) intersite cooperativity occurring within DNA-induced heterotetramers is equal to the average of the intersite cooperativity within a PR-A or PR-B saturated PRE₂. Representative ligation states and the ratio of PR-A to PR-B in the PR–PRE₂ complex are indicated in the schematics.