Structural proteomics defines a sequential priming mechanism for the progesterone receptor

Abstract

The progesterone receptor (PR) is a steroid-responsive nuclear receptor with two isoforms: PR-A and PR-B. Disruption of PR-A:PR-B signaling is associated with breast cancer through interactions with oncogenic co-regulatory proteins (CoRs). However, molecular details of isoform-specific PR-CoR interactions remain poorly understood. Using structural mass spectrometry, we investigate the sequential binding mechanism of purified full-length PR and intact CoRs, steroid receptor coactivator 3 (SRC3) and p300, as complexes on target DNA. Our findings reveal selective CoR NR-box binding by PR and unique interaction surfaces between PR and CoRs during complex assembly, providing a structural basis for CoR sequential binding on PR. Antagonist-bound PR showed persistent CoR interactions, challenging the classical model of nuclear receptor activation and repression. In this work, we offer a peptide-level perspective on the organization of the PR transcriptional complex and infer the mechanisms behind the interactions of these proteins, both in active and inactive conformations.

Fig. 1. DNA induces assembly of PR-A and PR-B.

a, b SEC-MALS chromatograms of agonist (R5020)- bound PR-A (a) and PR-B (b) with and without DNA. The molar mass of DNA and PR-A alone matches the monomeric molar mass (black line/dots across the peaks). DNA induces assembly of both PR-A and PR-B into a complex with 2:1 (protein:DNA) stoichiometry. The presence of DNA in the complexes was confirmed by deconvolution of the protein and DNA fractions in the peak (red and blue lines, respectively). Source data are provided as a Source Data file.

Fig. 2. Structural proteomics reveals isoform differences upon progesterone response element (PRE) binding.

A Consolidated HDX-MS data, run in triplicate, showing the differential analysis between unbound PR vs. PRE-bound, where the top is PR-B, and the bottom is PR-A. Domains are labeled as follows: N-terminal Domain (NTD), DNA-binding domain (DBD), Hinge region (Hinge), and Ligand-binding domain (LBD). Exchange data is representative of a full seven-timepoint differential HDX experiment with sample injection after 10, 30, 60, 300, 900, and 3600 s of deuterium exchange. B Trimmed AlphaFold 3.0 model (residues 375–769) of PR-A homodimer with unbound PR-A vs. PR-A:PRE HDX overlays. Highlighted regions are the PR dimerization domain (residues 885–922 within LBD) and the DBD C-terminal extension (right, residues 633–670). Residue labeling corresponds to the PR-B numbering. Cooler colors indicate comparative reductions in deuterium exchange. C XiView images of differential PR-A ± PRE experiments, where all validated crosslinks are shown. Selected N-terminal crosslinks not identified in PR-A:PRE XL-MS experiments are highlighted in red, with crosslinks mapped onto PR-B numbering, with the gray region representing the 164 amino acids not expressed in PR-A. Results representative of triplicate experiments, with validation in Skyline. D XiView of differential PR-B ± PRE experiments, where all validated crosslinks are shown. Selected N-terminal crosslinks not identified in PR-B:PRE XL-MS experiments are shown in red.

Fig. 3. SRC3 induces LBD changes to PR upon PRE addition.

A Left HDX overlay (PR-A vs. PR-A:SRC3, both non-DNA bound) mapped onto AlphaFold3.0 model of the PR-A:SRC3 ternary complex with the PR homodimer highlighted. Zoomed-in sections of PR corresponding to the dimerization domains (PR-B amino acids: 855-922 and 602-618) and N-terminal domain (PR-A amino acids 1-476) highlighted with matching HDX overlays. Right. Differential HDX overlay of SRC3 vs. PR-A:SRC3 onto the best scoring PR:SRC3 apo complex with SRC3 highlighted. NR-boxes 1 and 2 (amino acids 685-689 and 738-742, respectively) zoomed-in up to show differential exchange. B Left. HDX overlay (PR-A:PRE vs. PR-A:SRC3:PRE, both DNA-bound) mapped onto AlphFold3.0 model of PR-A:SRC3:PRE ternary complex with the PR homodimer highlighted. One PR-A monomer is shown as a zoomed-in section. B Right. Differential HDX overlay of SRC3 vs. PR-A:SRC3:PRE onto the best-scoring PR:SRC3 apo complex with SRC3 highlighted. NR-boxes 1 and 2 and the p300 interaction site (amino acids 1023-1093) are highlighted to show differential exchange. Black peptide regions correspond to peptides not identified by HDX-MS. Each color represents the percent change in deuterium incorporation (Δ%D), following the scale shown at the bottom, where darker blues correspond to decreased differential deuterium exchange and warmer reds correspond to increases in differential deuterium exchange. Exchange data is representative of a full seven-timepoint differential HDX experiment with sample injection after 10, 30, 60, 300, 900, and 3600 s of exchange time.

Fig. 4. p300 differentially alters the conformational dynamics of PR-A and PR-B within the PR:SRC3:p300 complex.

A Consolidated HDX plots of PR-A showing the differential HDX-MS comparisons within the plot to the left. Changes in deuterium uptake are represented by the rainbow plot shown, where darker blues correspond to decreased differential deuterium exchange and warmer reds correspond to increases in differential deuterium exchange. Common PR domains are highlighted above consolidated data: N-terminal domain (NTD, orange), DNA-binding domain (DBD, purple), Hinge (yellow), and ligand-binding domain (LBD, teal). B AlphaFold3.0 models of PR from the AF1 to LBD (amino acids 456-933 using PR-B numbering). HDX-MS overlays represent the same experiments as the consolidated views in A. Each color represents the percent change in deuterium incorporation (Δ%D), following the scale shown at the bottom. Exchange data is representative of a full seven-timepoint differential HDX experiment with sample injection after 10, 30, 60, 300, 900, and 3600 s of deuterium exchange. Gray overlays indicate no significant changes, and black indicates peptides not detected in the HDX-MS experiment.

Fig. 5. PR-A and PR-B differentially interact with SRC3 and are stabilized by p300 addition.

A. Consolidated differential HDX-MS results for SRC3, comparing the changes induced by PR-A and p300 binding in the presence and absence of PRE DNA. B Consolidated HDX-MS plot of SRC3 exchange, with PR-B comparisons in the same order as PR-A. The motifs highlighted are the following: bHLH (orange), PAS (purple), LXXLL motifs (yellow), CREBBP Interaction Domain (teal), and acetyltransferase domain (dark blue). Each color represents the percent change in deuterium incorporation (Δ%D), following the scale shown, where darker blues correspond to decreased differential deuterium exchange and warmer reds correspond to increases in differential deuterium exchange. Gray overlays indicate no significant changes, and black indicates peptides not detected in the HDX-MS experiment. C Selected deuterium uptake plots for peptides that contain LXXLL motifs 1, 2, and 3. The %D uptake indicates the percent deuterium uptake over time for the PR-A:SRC3 ± DNA and PR-A:SRC3:p300 ± DNA HDX experiments. Data points are the mean of three replicates (N = 3) with error bars corresponding to the standard deviation in the differential deuterium uptake for each time point. Statistics were derived using two-way ANOVA with Tukey post hoc correction for multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Exchange data is representative of a full seven-timepoint differential HDX experiment with sample injection after 10, 30, 60, 300, 900, and 3600 s of deuterium exchange.

Fig. 6. XL-MS shows p300 directly interacts with PR.

Crosslinking results from PR-B:p300±PRE experiments. Purple: intraprotein crosslinks, green: interprotein crosslinks. PR highlighted domains: DBD (purple) and LBD (green). p300 highlighted domains: bromodomain (pink), zinc finger domain (green), and NCOA2-interaction domain (yellow).

Fig. 7. RU486 antagonism rearranges PR-SRC3-p300 interactions.

A Plotted differential crosslinks in PR-B:SRC3 experiments, comparing R5020-specific (agonist, red) and RU486-specific (antagonist, blue) crosslinks. The x-axis represents the Log2 transformed fold change values from Skyline, while the y-axis represents the -log10 transformation of the Skyline p-value output. The lines are indicative of a Log2 fold change of 1 (two-fold increase) and -log10 p-value of 1.3, corresponding to p < 0.05. Each point represents a unique crosslink with corresponding XlinkX scores represented as point size. P-values were calculated by pairwise-ratio comparisons of the transition peak areas for crosslinked peptides in Skyline using linear mixed-effects models within the MSStats group comparison node. A Red. XlinkX view of R5020-specific crosslinks in differential PR:SRC3 experiments. A Blue. XlinkX view of RU486-specific crosslinks in differential PR:SRC3 experiments. B All validated R5020-bound crosslinks for differential PR-A:SRC3:p300 ± PRE (Left) and PR-B:SRC3:p300 ± PRE (Right) experiments. Selected crosslinks, highlighted in red, show PR: SRC3-specific crosslinks. C All validated RU486-specific crosslinks for the same experimental setup described in B. Red denotes all PR:SRC3 crosslinks detected with an XlinkX score ≥ 50. Defined domains are as follows: PR - DBD (purple) and LBD (green); SRC3 – NR-boxes (gold) and histone acetyltransferase domain (violet); p300 - bromodomain (pink), zinc finger domain (green), and NCOA2-interaction domain (yellow).

Fig. 8. RU486-bound PR has reduced deuterium exchange upon CoR binding.

A PR models of AF1 to C-terminus (amino acids 456-933) with PR-A HDX overlays, corresponding to the comparisons shown beneath them. B PR models of the AF1 to C-terminus with corresponding PR-B HDX overlays labeled beneath. Each color represents the percent change in deuterium incorporation (Δ%D), following the scale shown at the bottom. Gray overlays indicate no significant changes, and black indicates peptides not detected in the HDX-MS experiment. Exchange data is representative of a full seven-timepoint differential HDX experiment with sample injection after 10, 30, 60, 300, 900, and 3600 s of deuterium exchange.