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

Abstract

The anaphase-promoting complex/cyclosome (APC/C) is a large multimeric cullin–RING E3 ubiquitin ligase that orchestrates cell-cycle progression by targeting cell-cycle regulatory proteins for destruction via the ubiquitin proteasome system. The APC/C assembly comprises two scaffolding subcomplexes: the platform and the TPR lobe that together coordinate the juxtaposition of the catalytic and substrate-recognition modules. The platform comprises APC/C subunits Apc1, Apc4, Apc5, and Apc15. Although the role of Apc1 as an APC/C scaffolding subunit has been characterized, its specific functions in contributing toward APC/C catalytic activity are not fully understood. Here, we report the crystal structure of the N-terminal domain of human Apc1 (Apc1N) determined at 2.2-Å resolution and provide an atomic-resolution description of the architecture of its WD40 (WD40 repeat) domain (Apc1WD40). To understand how Apc1WD40 contributes to APC/C activity, a mutant form of the APC/C with Apc1WD40 deleted was generated and evaluated biochemically and structurally. We found that the deletion of Apc1WD40 abolished the UbcH10-dependent ubiquitination of APC/C substrates without impairing the Ube2S-dependent ubiquitin chain elongation activity. A cryo-EM structure of an APC/C–Cdh1 complex with Apc1WD40 deleted showed that the mutant APC/C is locked into an inactive conformation in which the UbcH10-binding site of the catalytic module is inaccessible. Additionally, an EM density for Apc15 is not visible. Our data show that Apc1WD40 is required to mediate the coactivator-induced conformational change of the APC/C that is responsible for stimulating APC/C catalytic activity by promoting UbcH10 binding. In contrast, Ube2S activity toward APC/C substrates is not dependent on the initiation-competent conformation of the APC/C.

Ubiquitin chain-elongating enzyme UBE2S activates the RING E3 ligase APC/C for substrate priming

“…One possible explanation for this result is that UBE2S CTP -dependent activation mimics coactivator-induced activation where a conformational change in the catalytic core allows for UBE2C recruitment. Previous studies revealed that the N-terminal WD40 domain of APC1 is necessary for this coactivator-dependent conformational change 26 . These studies show that deletion of the WD40 domain (APC/C ΔAPC1-WD40 ) locks the APC2–APC11 catalytic core in a “downward” conformation similar to the apo state rendering the UBE2C-binding site inaccessible in the presence of coactivator ( Fig.…”

“… a , Cartoon representing APC/C ΔAPC1-WD40 showing the catalytic core locked in a downward position and inaccessible for UBE2C recruitment, based on previous studies 26 . b , Substrate ubiquitination reactions comparing the effects of UBE2S CTP and UBES CTP−4A (residues 219-222 of UBE2S CTP substituted for alanine) on CycB NTD * priming, multiubiquitination, and Ub chain elongation by APC/C ΔAPC1-WD40 , UBE2C, and UBE2S.…”

“…This mobilizes the catalytic module to expose the UBE2C-binding site and facilitates UBE2C activation for ubiquitination 19 , 24 , 25 . The WD40 domain of the scaffolding subunit APC1 (APC1 WD40 ) is critical for mediating this conformational change; deletion of APC1 WD40 has been shown to lock the catalytic module in the downward position, blocking the binding site of UBE2C while leaving UBE2S binding unaffected 26 .…”

CDH1 regulates E2F1 degradation in response to differentiation signals in keratinocytes

“…The ubiquitylation of a substrate not only depends on its binding to Cdh1, but also on its relative position and proximity to the K11-linking machinery [39]. We determined that both wild type and ST/A E2F1 bind to Cdh1.…”

Mutations in ANAPC1, Encoding a Scaffold Subunit of the Anaphase-Promoting Complex, Cause Rothmund-Thomson Syndrome Type 1

“…Analysis by real-time cell cycle microscopy revealed a significant delay within the interphase for synchronized fibroblast cells derived from individuals with the intronic splicing mutation compared to control fibroblasts ( Figure 3A). There 24,25 The WD40 domain mediates conformational changes important for stimulating the APC's catalytic activity upon co-activator binding. The Mid-N and Mid-C domains coalesce together in the APC complex.…”