The mono-ubiquitination of the histone protein H2B (H2Bub1) is a highly conserved histone post-translational modification that plays critical roles in many fundamental processes. In yeast, this modification is catalyzed by the conserved Bre1–Rad6 complex. Bre1 contains a unique N-terminal Rad6-binding domain (RBD), how it interacts with Rad6 and contributes to the H2Bub1 catalysis is unclear. Here, we present crystal structure of the Bre1 RBD–Rad6 complex and structure-guided functional studies. Our structure provides a detailed picture of the interaction between the dimeric Bre1 RBD and a single Rad6 molecule. We further found that the interaction stimulates Rad6’s enzymatic activity by allosterically increasing its active site accessibility and likely contribute to the H2Bub1 catalysis through additional mechanisms. In line with these important functions, we found that the interaction is crucial for multiple H2Bub1-regulated processes. Our study provides molecular insights into the H2Bub1 catalysis.

Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt a biochemically defined off-pathway ‘deactive’ state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here, we report the structure of mitochondrial complex I from the thoracic muscles of the model protostome Drosophila melanogaster. We show that although D. melanogaster complex I (Dm-CI) does not have a NEM-sensitive deactive state, it does show slow activation kinetics indicative of an off-pathway resting state. The resting-state structure of Dm-CI from the thoracic muscle reveals multiple conformations. We identify a helix-locked state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of the Dm-CI structure and conformational states to those observed in bacteria, yeast, and mammals provides insight into the roles of subunits across organisms, explains why the Dm-CI off-pathway resting state is NEM insensitive, and raises questions regarding current mechanistic models of complex I turnover.