Mechanism of Polyubiquitination by Human Anaphase-Promoting Complex: RING Repurposing for Ubiquitin Chain Assembly

Abstract

Polyubiquitination by E2 and E3 enzymes is a predominant mechanism regulating protein function. Some RING E3s, including Anaphase Promoting Complex/Cyclosome (APC), catalyze polyubiquitination by sequential reactions with two different E2s. An initiating E2 ligates ubiquitin to an E3-bound substrate. Another E2 grows a polyubiquitin chain on the ubiquitin-primed substrate through poorly defined mechanisms. Here we show that human APC’s RING domain is repurposed for dual functions in polyubiquitination. The canonical RING surface activates an initiating E2~ubiquitin intermediate for substrate modification. However, APC engages and activates its specialized ubiquitin chain elongating E2 UBE2S in ways that differ completely from current paradigms. During chain assembly, a distinct APC11 RING surface helps deliver a substrate-linked ubiquitin to accept another ubiquitin from UBE2S. Our data define mechanisms of APC/UBE2S-mediated polyubiquitination, reveal unexpectedly diverse functions of RING E3s and E2s, and provide a framework for understanding distinctive RING E3 features specifying ubiquitin chain elongation.

Ube2s regulates Sox2 stability and mouse ES cell maintenance

“…Recently, two studies dissected the detailed mechanism used by Ube2s and its E3 APC to drive polyubiquitin chain formation on substrates. 54,55 In their model, APC simultaneously associates with Ube2s and substrate, and continuously adds Ub to the distal end of the growing polyubiquitin chain in a one-by-one manner. We speculate that the high affinity between Ube2s and Sox2 may enhance the efficiency of polyubiquitin chain formation on Sox2.…”

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes

“…These approaches typically require the generation of large sets of DNA constructs to identify tagged proteins that are suitable for purification or localization studies without perturbing protein function or to identify mutants that are defective in specific properties. For example, a comprehensive structure-function analysis of the ubiquitinconjugating enzyme UBE2S required the generation of 135 mutants, even though this monomeric protein is only composed of 222 amino acid residues (8). The application of tagging and mutagenesis approaches for the generation and analysis of multisubunit protein complexes is more difficult.…”

WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity

“…This LR tail-binding site does not change conformation on interconversion between active and inactive states (12,39). Although independent of the catalytic module for APC/C binding, the catalytic activity of Ube2S requires the RING domain of Apc11 that is repurposed to engage the acceptor ubiquitin of the APC/C substrate for covalent linkage with the donor ubiquitin of the Ube2S-ubiquitin conjugate (14,30). The acceptor ubiquitin-binding site on Apc11 is accessible in the inactive APC/C conformation ( Fig.…”

“…The work raises the possibility that conformational changes of the Apc1 WD40 domain may play a role in regulating UbcH10 binding to the APC/C. position the acceptor ubiquitin, conjugated to an APC/C substrate, for modification by Ube2S (14,30,31). UbcH10 and Ube2S together build branched ubiquitin chains on APC/C substrates, and these chains are recognized more efficiently by the proteasome (32).…”

“…The association of UbcH10 with the RING domain of Apc11 (Apc11 RING ) and Apc2's winged-helix B domain (Apc2 WHB ) initiates ubiquitin-chain formation (19,20,(24)(25)(26). Ube2S, on the other hand, is responsible for ubiquitin-chain extension and specifically assembles Lys11-linked ubiquitin chains on substrates targeted by the APC/C (14,25,(27)(28)(29)(30). In vertebrates, the RING domain of Apc11 is repurposed to…”