R01CA263574

Structural Dynamics of Progesterone Receptor-Coactivator Complexes - Summary

Steroid hormone receptors (SR) are ligand-dependent nuclear transcription factors that exhibit remarkable functional diversity in mediating cell/tissue and target gene specific responses. This diversity is largely driven by the conformational dynamics of the SR protein, which enables its binding to unique subsets of transcriptional co-regulatory proteins (CoRs) and DNA response elements.

The progesterone receptor (PR) is the main target of progestogens that are widely used clinically. PR is expressed as two protein isoforms, an N-terminal truncated PR-A and full-length PR-B, each having distinct physiological roles dependent on the cell/tissue type. Generally, PR-A is a weaker transcriptional activator than PR-B and can act to attenuate the activity of PR-B. Both isoforms are typically co-expressed in equal proportions in most normal tissues. However, PR-A to PR-B ratios have been reported to be highly variable in pathological conditions. The mechanistic basis for differences in activity between the isoforms is not well defined but is generally believed to be due to unknown differences in structural conformations.

To fully understand PR's biology, it is necessary to determine a high-resolution structure of the full-length PR isoforms and associated CoRs as a complex on target DNA, and to understand how protein interactions within the complex and structural conformations affect PR activity. However, the conformational flexibility of SRs and CoRs, coupled with their large sizes (100-300 MW), make them unsuitable for high-resolution NMR or X-ray crystallography analysis.

As an alternative, this proposal aims to integrate complementary solution-phase techniques to determine high-resolution 3D structural models and uncover the conformational dynamics within the PR:CoR/DNA complex. Recent advances in cryo-EM enable the determination of solution-phase structures of large conformationally heterogeneous macromolecular complexes at subnanometer resolution. In this project, cryo-EM will be combined with crosslinking mass spectrometry (XL-MS) to further refine structural cryo-EM models and assure high resolution. Hydrogen-deuterium exchange (HDX) will be used to map conformational dynamics and allostery within the PR:CoR/DNA complex.

The overall goal of this project is to determine the highest resolution 3D structure possible of full-length PR-A and PR-B in complex with classical CoRs and novel CoRs on PR DNA response elements. Aim 1 will utilize cryo-EM to analyze the structural features of PR-A and PR-B in complex with the classical CoRs SRC3 and p300, and with the novel CoRs TBP and JDP2 assembled on target DNA. Aim 2 will refine the cryo-EM structure of the PR:CoR/DNA complex using integrated structural modeling and XL-MS to define distance constraints and probe conformational dynamics within the PR complex by differential HDX. Aim 3 will perform functional mutagenesis studies to determine the influence of PR:SRC3/p300 interaction surfaces revealed in structural models and from XL-MS data on PR activity.

The impact of this proposal will be to fill a major gap in our understanding of the structure and conformational dynamics of the PR:CoR/DNA complex. These studies could open opportunities for novel studies of drug interactions at the atomic level.

University Of Florida

NOT APPLICABLE

93.396 - Cancer Biology Research

National Cancer Institute (NCI) [HHS - NIH]

Jupiter, Florida 33458 United States

Single Zip Code

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 365% from $655,489 to $3,046,984.