Tuesday, April 29, 2025
4/29/2025
Structure of the BRCA1-PALB2-BRCA2 complex - PROJECT SUMMARY The BRCA1, PALB2, and BRCA2 proteins form a heterocomplex that is involved in the maintenance of genomic stability, and variants of these proteins are implicated in multiple cancers. Unfortunately, the full-length structural details for BRCA1, PALB2, and BRCA2 are unsolved which obscures a complete understanding of their molecular mechanism of action. Our long-term goal is to understand the molecular features of the BRCA1- PALB2-BRCA2 axis and how disease related mutants impact their structure such that better targeted treatment approaches can be realized. The primary goal of this proposal is to produce full-length wild type BRCA1, PALB2, and BRCA2 (along with additional protein partners BARD1, DSS1 and RAD51) to determine the structure of stabilized heterocomplexes. Our hypothesis is that the past difficulty in solving the structure of these core proteins is due to the presence of intrinsically disordered domains that fail to adopt a stable conformation unless part of a larger heterocomplex. The rationale and innovation underlying the proposed work is that cell-free expression allows small scale, versatile and rapid optimization of conditions to increase the chances of synthesizing a full-length protein with similar solubility, biochemical and other characteristics as the native protein. Additionally, cryogenic electron microscopy (cryo-EM) can interrogate proteins that adopt multiple conformations or contain flexible or intrinsically disordered regions as BRCA1, PALB2 and BRCA2 are known to possess. Two specific aims will be pursued to complete this work: 1) Express, purify, and characterize BRCA1, PALB2, and BRCA2 complexes. 2) Determine structure of BRCA1, PALB2, and BRCA2 complexes using single particle cryo-EM. The success of the project will hinge on recent advancements in cryo-EM and our cell- free expression pipeline. The proposed research is significant since it will provide a flexible framework for production and structural characterization of BRCA1, PALB2, and BRCA2 complexes. These aims directly align with the funding opportunity NIH PAR-23-058 “NCI Small Grants Program for Cancer Research for Years 2023, 2024, and 2025” as our goal is to generate an improved understanding of multiple tumor suppressor proteins. This basic research proposal is both a feasibility study, as well as a small self-contained research project that can be completed in two years. More in-depth molecular phenotyping, and functional and mechanistic insights into BRCA-related DNA repair processes are beyond the scope of the current proposal due to both timeframe and budget. That work in combination with more detailed structural comparison of more disease related variants would be the focus of a separate follow-on proposal. The expected outcome of this work is the synthesis of wild type BRCA1, PALB2, and BRCA2 along with structural characterization of the BRCA1- PALB2-BRCA2 axis. It will also lay the foundation for a workflow extending our cell-free expression pipeline from bioenergy research into the biomedical field to permit its application toward other cancer related proteins whose structure remain elusive.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).