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Title: Capturing a substrate in an activated RING E3/E2–SUMO complex

Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
Resource Type:
Journal Article
Resource Relation:
Journal Name: Nature (London); Journal Volume: 536; Journal Issue: 7616
Country of Publication:
United States

Citation Formats

Streich Jr, Frederick C., and Lima, Christopher D.. Capturing a substrate in an activated RING E3/E2–SUMO complex. United States: N. p., 2016. Web. doi:10.1038/nature19071.
Streich Jr, Frederick C., & Lima, Christopher D.. Capturing a substrate in an activated RING E3/E2–SUMO complex. United States. doi:10.1038/nature19071.
Streich Jr, Frederick C., and Lima, Christopher D.. 2016. "Capturing a substrate in an activated RING E3/E2–SUMO complex". United States. doi:10.1038/nature19071.
title = {Capturing a substrate in an activated RING E3/E2–SUMO complex},
author = {Streich Jr, Frederick C. and Lima, Christopher D.},
abstractNote = {},
doi = {10.1038/nature19071},
journal = {Nature (London)},
number = 7616,
volume = 536,
place = {United States},
year = 2016,
month = 8
  • How RING E3 ligases mediate E2-to-substrate ubiquitin-like protein (UBL) transfer remains unknown. Here we address how the RING E3 RBX1 positions NEDD8's E2 (UBC12) and substrate (CUL1). We find that existing structures are incompatible with CUL1 NEDD8ylation and report a new conformation of RBX1 that places UBC12 adjacent to CUL1. We propose RING domain rotation as a general mechanism for UBL transfer for the largest family of E3s.
  • 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 collaboratesmore » 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. In this paper, we report structural mechanisms of UBCH10 recruitment to APC CDH1 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 APC CDH1–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. Finally, 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.« less
  • Smc5/6, a protein complex that belongs to the structural maintenance of chromosome (SMC) family, plays a key role in DNA replication, sister chromatid recombination and DNA damage repair. The complex contains eight subunits, including a SUMO E3 ligase Mms21 (Nse2). The activity of Mms21 is important for regulation of Smc5/6 in the response to DNA damage. Mms21 and the Mms21-binding region of Smc5 were overexpressed and purified individually in Escherichia coli with a C-terminal LEHHHHHH tag. The Mms21-Smc5 protein complex was crystallized. The diffraction of the crystals was improved greatly by glutaraldehyde treatment. X-ray diffraction data sets were collected tomore » resolutions of 2.3 and 3.9 {angstrom} from native and selenomethionine-derivative protein crystals, respectively. The crystals belonged to space group C2221, with unit-cell parameters a = 47.465, b = 97.574, c = 249.215 {angstrom} for the native crystals.« less