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Title: RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex

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. In this paper, 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 tripartitemore » 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
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [1] ;  [1] ;  [4] ;  [1] ;  [5] ;  [1] ;  [6] ;  [7] ;  [6] ;  [4] ;  [5] ;  [8]
  1. St. Jude Children's Research Hospital, Memphis, TN (United States). Dept. of Structural Biology
  2. St. Jude Children's Research Hospital, Memphis, TN (United States). Dept. of Structural Biology. Howard Hughes Medical Inst.
  3. St. Jude Children's Research Hospital, Memphis, TN (United States). Dept. of Structural Biology; Univ. of Tennessee Health Sciences Center, Memphis, TN (United States). Dept. of Microbiology, Immunology, and Biochemistry
  4. Vienna Biocenter (VBC) (Austria). Research Inst. of Molecular Pathology (IMP)
  5. Max Planck Inst. for Biophysical Chemistry, Gottingen (Germany); Univ. of Gottingen (Germany). Dept. of 3D Electron Cryomicroscopy. Inst. of Microbiology and Genetics
  6. St. Jude Children's Research Hospital, Memphis, TN (United States). Dept. of Chemical Biology and Therapeutics
  7. St. Jude Children's Research Hospital, Memphis, TN (United States). Hartwell Center for Bioinformatics and Biotechnology
  8. St. Jude Children's Research Hospital, Memphis, TN (United States). Dept. of Structural Biology. Howard Hughes Medical Inst.; Univ. of Tennessee Health Sciences Center, Memphis, TN (United States). Dept. of Microbiology, Immunology, and Biochemistry
Publication Date:
OSTI Identifier:
1197027
Grant/Contract Number:
AC02-06CH11357; R37GM065930; P30CA021765; P41 GM103403; RSG CDD-120969; 227764 MitoSys; Sonderforschungsbereich 860
Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 112; Journal Issue: 17; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
St. Jude Children's Research Hospital, Memphis, TN (United States); Vienna Biocenter (VBC) (Austria); Max Planck Inst. for Biophysical Chemistry, Gottingen (Germany); Univ. of Gottingen (Germany)
Sponsoring Org:
USDOE; National Inst. of Health (NIH) (United States); Jane Coffin Childs Foundation (United States); American Cancer Society (United States); American Lebanese Syrian Associated Charities (ALSAC) (United States); Howard Hughes Medical Inst. (United States); Japan Society for the Promotion of Science (Japan); Boehringer Ingelheim (Germany); Laura Bassi Centre for Optimized Structural Studies (Austria); European Union (Belgium); Austrian Research Fund (Austria); German Research Foundation (DFG)
Contributing Orgs:
Univ. of Tennessee Health Sciences Center, Memphis, TN (United States)
Country of Publication:
United States
Language:
ENGLISH
Subject:
60 APPLIED LIFE SCIENCES; ubiquitin; anaphase-promoting complex; cullin; E3; electron microscopy