Steric Gating Contributes to the Preferential Ubiquitin Charging of BIRC6 by UBA6 versus UBA1

The giant E2-E3 chimera BIRC6 specifically engages ubiquitin-activating E1 enzyme UBA6, but not UBA1, enabling UBA6 to transfer activated ubiquitin to BIRC6, which then ubiquitinates downstream substrates such as caspases to regulate apoptosis. However, the molecular mechanism that underlies UBA6-mediated specific engagement of BIRC6 remains elusive. Here we use a Ub^Dha probe to trap the transient E1-E2 transthioesterification intermediate and determine cryo-EM structures of singly and doubly loaded UBA6-BIRC6^UBC complexes at 3.3 and 3.4 Å resolution. Structural analysis reveals that an insertion loop (residues 4649-4653) in BIRC6 and the gate helix of UBA6 play a key role in the specific BIRC6-UBA6 pairing. Unlike UBA1, UBA6 avoids steric clash through a ~ 30° rotation and a 19 Å displacement of its gate helix, creating a compatible cavity for the insertion loop. Biochemical validation confirms that truncation of the BIRC6 insertion loop rescues UBA1‑mediated charging, and swapping the UBA6 gate helix into UBA1 confers activity toward BIRC6^UBC. Our study provides structural insights into E1-E2 pairing specificity and advances the mechanistic understanding of apoptotic signaling.