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

doi:10.1073/pnas.0608848104

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

Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, CO 80262

Edited by Jan-Åke Gustafsson, Karolinska Institutet, Huddinge, Sweden, and approved December 14, 2006 (received for review October 5, 2006)

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.

Footnotes

*To whom correspondence should be addressed at:
Department of Pharmaceutical Sciences, C-238, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262.
E-mail: david.bain{at}uchsc.edu
Author contributions: D.L.B. designed research; K.D.C.-J., A.F.H., and M.T.M. performed research; K.D.C.-J. and A.F.H. analyzed data; and K.D.C.-J., A.F.H., and D.L.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
↵ † Rigorous thermodynamic analysis of the MMTV promoter indicates that neither PR-B nor PR-A can engage in cooperative interactions between the MMTV palindromic site and the half-sites (A.F.H. and D.L.B., unpublished work).
Abbreviations:

DBD,

DNA binding domain;

PR,

progesterone receptor;

PRE,

progesterone response element;

PR-A,

A-isoform of PR;

PR-B,

B-isoform of PR;

BUS,

B-unique sequence;

PRE2,

DNA template containing two PREs;

PRE1−,

DNA template containing a single PRE.