Small Structural Variations, Large Functional Consequences: Comparative Analysis Reveals Structural Control of...

BRCA1/BARD1 is a chromatin-associated E3 ubiquitin ligase that ubiquitylates histone H2A to coordinate DNA damage repair, transcriptional repression, and genome stability. In Caenorhabditis elegans (C. elegans), the orthologous BRC-1/BRD-1 complex performs analogous functions but exhibits structural variation, most notably through an additional 11-residue loop in BRD-1 that is absent from human BARD1. Prior experiments indicate this worm-specific insertion promotes nucleosome engagement and may alter the preferred lysine target for ubiquitylation. Here, we provide a cross-species comparison by integrating computational and experimental investigation to clarify how a discrete structural variation can tune BRCA1-family ligase behavior and, consequently, chromatin regulation. In vitro ubiquitylation assays and mass spectrometry reveal BRC-1/BRD-1 ubiquitylate the C-terminal tail of histone H2A with less specificity than the human homologs. All-atom molecular dynamics simulations of both the C. elegans BRC-1/BRD-1-LET-70-Ubiquitin assembly and the human BRCA1/BARD1-UbcH5c-Ubiquitin complex in the presence of the nucleosome core particle uncover that the BRD-1 loop makes transient contacts with nucleosomal DNA and histone tails, thereby modulating the positioning and conformational flexibility of the bound E2 (ubiquitin-conjugating enzyme). Together, our results suggest that the BRD-1 loop alters the E3-E2 geometry, thereby altering ubiquitylation-site specificity.