Structural proteomics defines a sequential priming mechanism for the progesterone receptor.

The progesterone receptor (PR) is a steroid-responsive nuclear receptor, expressed as two isoforms: PR-A and PR-B. The isoforms display distinct expression patterns and biological actions in reproductive target tissues and disruption of PR-A:PR-B signaling is associated with breast cancer development potentially by altering interactions with oncogenic co-regulatory protein (CoRs). However, the molecular details of isoform-specific PR-CoR interactions that influence progesterone signaling remain poorly understood. We employed structural mass spectrometry in this study to investigate the sequential binding mechanism of purified full-length PR and full-length CoRs, steroid receptor coactivator 3 (SRC3) and p300, as complexes with target DNA. Our findings reveal selective CoR NR-box binding by PR and novel interaction surfaces between PR, SRC3, and p300, which change during complex assembly. This provides a structural model for a sequential priming mechanism that activates PR. Comparisons of PR bound to progesterone agonist versus antagonist challenges the classical model of nuclear receptor activation and repression. Collectively, we offer a peptide-level perspective on the organization of the PR transcriptional complex and elucidate the mechanisms behind the interactions of these proteins, both in active and inactive conformations.