Structural Oncology - Determining 3D Structures of Breast Cancer Assemblies

Abstract

Under normal cellular conditions, the breast cancer susceptibility protein (BRCA1) protects the genome by acting as a tumor suppressor. Cells harboring mutations in the BRCA1 gene lose the ability to properly repair DNA damage and transcribe their genome. These effects can contribute to genomic instability and cancer induction [1]. Indeed, mutations in the BRCA1 gene are heavily linked to the development of hereditary breast and ovarian cancers [2]. A major question in the field remains, how do mutations in BRCA1 disrupt molecular processes?Currently, there is little information available for how BRCA1 interacts with other proteins. Conventional biochemical separations do not yield BRCA1 complexes suitable for structural analysis. To address this issue, we have recently developed a tunable microchip system that facilitates the rapid recovery of native protein assemblies from the nuclear material of patient-derived breast cancer cells (Figure 1). We employed the new microchip system to isolate and visualize BRCA1-transcriptional complexes, for the first time. We collected cryo-EM images of BRCA1 assemblies under low-dose conditions and processed the images using the RELION software package [3]. Resulting 3D structures of the assemblies were interpreted by using a combination of antibody-labeling and molecular modeling techniques. This information allowed us to interpret the interactions between the RNAP II core complex and BRCA1 structural elements in the presence of DNA fragments. K63-linked ubiquitin moieties were also resolved in the structure, indicating a signal for DNA damage repair. Complementary biochemical experiments supported these findings to reveal the first 3D insights of BRCA1 protein assemblies in the context of human disease [4].Additional molecular modeling experiments on the BRCA1 C-terminal domain (BRCT) suggested a mechanism for peptide interactions within the BRCT binding site (Figure 2a, b). Phosphorylated peptide repeats present in the RNAP II core (pSer5) have the proper physical attributes to fit within the BRCT binding site. However, models of a prevalent BRCA1 clinical mutation (BRCA1 5382insC ) showed that the mutated BRCT domain does not support these associations (Figure 2c, d). Biochemical analysis of protein interactions with BRCA1 5382insC also indicated decreased affinity for its nuclear partner BARD1 (Figure 2e). Overall, these findings demonstrate new strategies to delineate the multifaceted role of BRCA1 in RNA metabolism while defining opportunities to dissect native BRCA1 protein interactions.