TIME-RESOLVED CRYO-EM ANALYSIS OF SUBSTRATE POLYUBIQUITINATION BY THE RING E3 ANAPHASE-PROMOTING COMPLEX/CYCLOSOME (APC/C)

Substrate polyubiquitination by the >600 ubiquitin ligases in the cell drives a myriad of cellular processes, including the cell cycle, apoptosis, and the immune response. The Anaphase Promoting Complex/Cyclosome (APC/C) is a 1.2 MDa RING E3 ubiquitin ligase that orchestrates the cell cycle by polyubiquitinating cell cycle regulators to mark them for proteasomal degradation. Several cofactors work with the APC/C to regulate its target specificity and the extent of ubiquitination, allowing the APC/C to dynamically control the cell cycle. Mechanistic insights into APC/C and other E3-mediated polyubiquitination have been obtained through elegant biochemical and structural studies using chemically trapped complexes to visualize snapshots of their catalytic activity. However, several questions remain about how an E3 builds a proteasomal degradation signal, which is regarded as 4 or more ubiquitins attached to a substrate, as the few available structures that mimic polyubiquitination are limited to di-ubiquitin formation. A complete picture of the process of polyubiquitination and how cofactors influence its conformational landscape remains a significant challenge. Here, we reconstituted APC/C-dependent polyubiquitination with its co-enzymes UBE2C and UBE2S for time-resolved cryo-EM. Using cryoDRGN (Deep Reconstructing Generative Networks), a neural network-based approach, we reconstruct the conformational changes undergone by the APC/C during polyubiquitination, directly visualize an active E3-E2 pair modifying its substrate, and identify unprecedented interactions between multiple substrate-linked ubiquitins with the APC/C’s coactivator and the E2 UBE2C. Together, we demonstrate how Ub modification of substrates on nascent Ub chains helps to potentiate processive substrate polyubiquitination and show how an E3 builds a polyubiquitinated substrate for proteasomal degradation.