Steric Gating Dictates Selective Activation of BIRC6 by UBA6 Over UBA1

The giant E2-E3 chimera BIRC6 is specifically activated by the ubiquitin-activating enzyme UBA6, but not by UBA1. This specificity allows BIRC6 to ubiquitinate downstream substrates (e.g., caspases) and thereby regulate apoptosis. However, the molecular mechanism that underlies UBA6mediated specific activation of BIRC6 remains elusive. Here, we used a UbDha (Ub residue 76 dehydroalanine) probe to achieve one-step, activity-based trapping of the transient transthioesterification intermediate, and resolved the cryo-EM structures of two key complexes: 1) the doubly loaded UBA6-BIRC6UBC-UbT-UbA complex (3.4 [A] resolution) and 2) the singly loaded UBA6-BIRC6UBC-UbT complex (3.3 [A] resolution). Structural analysis reveals that the specific BIRC6-UBA6 pairing is dictated by steric compatibility between an insertion loop (residues 4649- 4653) in BIRC6s UBC domain and UBA6s gate helix. Unlike UBA1, UBA6 avoids steric clash with this insertion loop via two key conformational changes: a ~30{degrees} clockwise rotation and a 19 [A] displacement of its gate helix. These conformational changes collectively create a compatible cavity to accommodate the insertion loop. Biochemical validation confirms two key findings: 1) truncating BIRC6s insertion loop rescues UBA1-mediated ubiquitin charging of BIRC6UBC; 2) swapping UBA6s gate helix with that of UBA1 abolishes UBA6s transthioesterification activity towards BIRC6UBC. Our study provides key structural insights into the molecular basis of E1-E2 pairing specificity and advances our mechanistic understanding of apoptotic signalling.