期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：17
页码：5272-5279
DOI：10.1073/pnas.1504161112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe anaphase-promoting complex/cyclosome (APC) is a multisubunit RING E3 ubiquitin (Ub) ligase that regulates mitosis, meiosis, and numerous facets of neurobiology by targeting key regulatory proteins for Ub-mediated degradation. Despite great importance, it remains unclear how APC, or most of the other 600 RING E3s in humans, targets Ub to lysines in disordered substrates. Here, we report the structural and molecular basis for substrate ubiquitination by APC and its partner E2, UBCH10. UBCH10 is recruited to APC, activated for ubiquitination, and positioned for substrate targeting through multisite interactions with the APC cullin-RING core. We propose that many RING E3-E2 assemblies work similarly, with multisite interactions establishing specificity, harnessing ubiquitination machineries to accelerate searching for target lysines, and facilitating regulation. For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2[~]Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING-E2[~]Ub catalytic modules such as APC11-UBCH10[~]Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APCCDH1 and substrate ubiquitination. Unexpectedly, in addition to binding APC11's RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APCCDH1-UBCH10[~]Ub-substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin-RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin-RING-E2 interactions establish APC's specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3-E2[~]Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.
