skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site

Abstract

Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub’s C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at “exosites.” However, identification of such sites is typically empirical and particularly challenging in large molecular machines. In this work, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The resultsmore » provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.« less

Authors:
 [1];  [2];  [3];  [2];  [4];  [5];  [2];  [6];  [6];  [5];  [7];  [8];  [9];  [10];  [3];  [1]
  1. St. Jude Children’s Research Hospital, Memphis, TN (United States); Max Planck Inst. of Biochemistry, Martinsried (Germany)
  2. St. Jude Children’s Research Hospital, Memphis, TN (United States)
  3. Univ. of Toronto, ON (Canada)
  4. Vienna BioCenter (Austria)
  5. Max Planck Inst. of Biochemistry, Martinsried (Germany)
  6. Univ. of North Carolina, Chapel Hill, NC (United States). Lineberger Comprehensive Cancer Center
  7. Vienna BioCenter (Austria); Max Planck Inst. for Biophysical Chemistry, Göttingen (Germany)
  8. Max Planck Inst. for Biophysical Chemistry, Göttingen (Germany)
  9. Vienna BioCenter (Austria); Medical Univ. of Vienna (Austria)
  10. St. Jude Children’s Research Hospital, Memphis, TN (United States); Univ. of North Carolina, Chapel Hill, NC (United States). Lineberger Comprehensive Cancer Center
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE; German Research Foundation (DFG); Austrian Research Promotion Agency; European Union (EU); Austrian Science Fund (FWF); National Institutes of Health (NIH); Genome Canada
OSTI Identifier:
1562701
Grant/Contract Number:  
860; 227764; SFB-F34; R35GM128855; R37GM065930; P30CA021765; OGI-119
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 116; Journal Issue: 35; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; anaphase-promoting complex; APC/C; ubiquitin; E3 ligase; inhibitor

Citation Formats

Watson, Edmond R., Grace, Christy R. R., Zhang, Wei, Miller, Darcie J., Davidson, Iain F., Prabu, J. Rajan, Yu, Shanshan, Bolhuis, Derek L., Kulko, Elizaveta T., Vollrath, Ronnald, Haselbach, David, Stark, Holger, Peters, Jan-Michael, Brown, Nicholas G., Sidhu, Sachdev S., and Schulman, Brenda A.. Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site. United States: N. p., 2019. Web. https://doi.org/10.1073/pnas.1902889116.
Watson, Edmond R., Grace, Christy R. R., Zhang, Wei, Miller, Darcie J., Davidson, Iain F., Prabu, J. Rajan, Yu, Shanshan, Bolhuis, Derek L., Kulko, Elizaveta T., Vollrath, Ronnald, Haselbach, David, Stark, Holger, Peters, Jan-Michael, Brown, Nicholas G., Sidhu, Sachdev S., & Schulman, Brenda A.. Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site. United States. https://doi.org/10.1073/pnas.1902889116
Watson, Edmond R., Grace, Christy R. R., Zhang, Wei, Miller, Darcie J., Davidson, Iain F., Prabu, J. Rajan, Yu, Shanshan, Bolhuis, Derek L., Kulko, Elizaveta T., Vollrath, Ronnald, Haselbach, David, Stark, Holger, Peters, Jan-Michael, Brown, Nicholas G., Sidhu, Sachdev S., and Schulman, Brenda A.. Fri . "Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site". United States. https://doi.org/10.1073/pnas.1902889116. https://www.osti.gov/servlets/purl/1562701.
@article{osti_1562701,
title = {Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site},
author = {Watson, Edmond R. and Grace, Christy R. R. and Zhang, Wei and Miller, Darcie J. and Davidson, Iain F. and Prabu, J. Rajan and Yu, Shanshan and Bolhuis, Derek L. and Kulko, Elizaveta T. and Vollrath, Ronnald and Haselbach, David and Stark, Holger and Peters, Jan-Michael and Brown, Nicholas G. and Sidhu, Sachdev S. and Schulman, Brenda A.},
abstractNote = {Ubiquitin (Ub)-mediated proteolysis is a fundamental mechanism used by eukaryotic cells to maintain homeostasis and protein quality, and to control timing in biological processes. Two essential aspects of Ub regulation are conjugation through E1-E2-E3 enzymatic cascades and recognition by Ub-binding domains. An emerging theme in the Ub field is that these 2 properties are often amalgamated in conjugation enzymes. In addition to covalent thioester linkage to Ub’s C terminus for Ub transfer reactions, conjugation enzymes often bind noncovalently and weakly to Ub at “exosites.” However, identification of such sites is typically empirical and particularly challenging in large molecular machines. In this work, studying the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which controls cell division and many aspects of neurobiology, we discover a method for identifying unexpected Ub-binding sites. Using a panel of Ub variants (UbVs), we identify a protein-based inhibitor that blocks Ub ligation to APC/C substrates in vitro and ex vivo. Biochemistry, NMR, and cryo-electron microscopy (cryo-EM) structurally define the UbV interaction, explain its inhibitory activity through binding the surface on the APC2 subunit that recruits the E2 enzyme UBE2C, and ultimately reveal that this APC2 surface is also a Ub-binding exosite with preference for K48-linked chains. The results provide a tool for probing APC/C activity, have implications for the coordination of K48-linked Ub chain binding by APC/C with the multistep process of substrate polyubiquitylation, and demonstrate the power of UbV technology for identifying cryptic Ub-binding sites within large multiprotein complexes.},
doi = {10.1073/pnas.1902889116},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 35,
volume = 116,
place = {United States},
year = {2019},
month = {7}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 3 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Cell Cycle-Regulated Recognition of the Destruction Box of Cyclin B by the APC/C in Xenopus Egg Extracts
journal, January 2004


A General Strategy for Discovery of Inhibitors and Activators of RING and U-box E3 Ligases with Ubiquitin Variants
journal, October 2017


E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin
journal, December 2013

  • Huang, Hao; Ceccarelli, Derek F.; Orlicky, Stephen
  • Nature Chemical Biology, Vol. 10, Issue 2
  • DOI: 10.1038/nchembio.1412

A Strategy for Modulation of Enzymes in the Ubiquitin System
journal, January 2013


Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation
journal, March 2011

  • Maspero, Elena; Mari, Sara; Valentini, Eleonora
  • EMBO reports, Vol. 12, Issue 4
  • DOI: 10.1038/embor.2011.21

Structure and function of a HECT domain ubiquitin-binding site
journal, March 2011

  • Kim, Hyung Cheol; Steffen, Alanna M.; Oldham, Michael L.
  • EMBO reports, Vol. 12, Issue 4
  • DOI: 10.1038/embor.2011.23

Identification of a physiological E2 module for the human anaphase-promoting complex
journal, October 2009

  • Williamson, A.; Wickliffe, K. E.; Mellone, B. G.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 43
  • DOI: 10.1073/pnas.0907887106

Conformational stabilization of ubiquitin yields potent and selective inhibitors of USP7
journal, November 2012

  • Zhang, Yingnan; Zhou, Lijuan; Rouge, Lionel
  • Nature Chemical Biology, Vol. 9, Issue 1
  • DOI: 10.1038/nchembio.1134

Enhanced Protein Degradation by Branched Ubiquitin Chains
journal, May 2014


Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation
journal, April 1996


Ubiquitin Ligases: Structure, Function, and Regulation
journal, June 2017


Multitasking with ubiquitin through multivalent interactions
journal, June 2010


Ubiquitin-binding domains
journal, August 2005

  • Hicke, Linda; Schubert, Heidi L.; Hill, Christopher P.
  • Nature Reviews Molecular Cell Biology, Vol. 6, Issue 8
  • DOI: 10.1038/nrm1701

Specificity of the anaphase-promoting complex: A single-molecule study
journal, April 2015


Molecular basis of APC/C regulation by the spindle assembly checkpoint
journal, August 2016

  • Alfieri, Claudio; Chang, Leifu; Zhang, Ziguo
  • Nature, Vol. 536, Issue 7617
  • DOI: 10.1038/nature19083

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15
journal, April 2019


Panta rhei: The APC/C at steady state
journal, April 2013

  • Primorac, Ivana; Musacchio, Andrea
  • The Journal of Cell Biology, Vol. 201, Issue 2
  • DOI: 10.1083/jcb.201301130

A Structure-Based Strategy for Engineering Selective Ubiquitin Variant Inhibitors of Skp1-Cul1-F-Box Ubiquitin Ligases
journal, September 2018


Ubiquitin Chain Elongation Requires E3-Dependent Tracking of the Emerging Conjugate
journal, October 2014


Structural insights into the catalysis and regulation of E3 ubiquitin ligases
journal, August 2016

  • Buetow, Lori; Huang, Danny T.
  • Nature Reviews Molecular Cell Biology, Vol. 17, Issue 10
  • DOI: 10.1038/nrm.2016.91

RBR ligase–mediated ubiquitin transfer: a tale with many twists and turns
journal, May 2018


System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes
journal, April 2016


Targeting HECT-type E3 ligases - insights from catalysis, regulation and inhibitors
journal, August 2017


Mechanism of Polyubiquitination by Human Anaphase-Promoting Complex: RING Repurposing for Ubiquitin Chain Assembly
journal, October 2014


Atomic structure of the APC/C and its mechanism of protein ubiquitination
journal, June 2015


Building a Regulatory Network with Short Linear Sequence Motifs: Lessons from the Degrons of the Anaphase-Promoting Complex
journal, October 2016


Ubiquitin-binding domains — from structures to functions
journal, October 2009

  • Dikic, Ivan; Wakatsuki, Soichi; Walters, Kylie J.
  • Nature Reviews Molecular Cell Biology, Vol. 10, Issue 10
  • DOI: 10.1038/nrm2767

Spatiotemporal regulation of the anaphase-promoting complex in mitosis
journal, January 2015

  • Sivakumar, Sushama; Gorbsky, Gary J.
  • Nature Reviews Molecular Cell Biology, Vol. 16, Issue 2
  • DOI: 10.1038/nrm3934

Structural mechanisms of HECT-type ubiquitin ligases
journal, January 2018


Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation
journal, August 2016


Activation of a Primed RING E3-E2–Ubiquitin Complex by Non-Covalent Ubiquitin
journal, April 2015


Molecular architecture and mechanism of the anaphase-promoting complex
journal, July 2014


Structure of ubiquitin refined at 1.8 Å resolution
journal, April 1987

  • Vijay-kumar, Senadhi; Bugg, Charles E.; Cook, William J.
  • Journal of Molecular Biology, Vol. 194, Issue 3
  • DOI: 10.1016/0022-2836(87)90679-6

UBE2S elongates ubiquitin chains on APC/C substrates to promote mitotic exit
journal, October 2009

  • Garnett, Mathew J.; Mansfeld, Jörg; Godwin, Colin
  • Nature Cell Biology, Vol. 11, Issue 11
  • DOI: 10.1038/ncb1983

A 20s complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B
journal, April 1995


UBE2S drives elongation of K11-linked ubiquitin chains by the Anaphase-Promoting Complex
journal, January 2010

  • Wu, T.; Merbl, Y.; Huo, Y.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 4
  • DOI: 10.1073/pnas.0912802107

Catalysis of Lysine 48-Specific Ubiquitin Chain Assembly by Residues in E2 and Ubiquitin
journal, August 2010


Assembly and Function of Heterotypic Ubiquitin Chains in Cell-Cycle and Protein Quality Control
journal, November 2017


Structural and Functional Insights to Ubiquitin-Like Protein Conjugation
journal, May 2014


RING-type E3 ligases: Master manipulators of E2 ubiquitin-conjugating enzymes and ubiquitination
journal, January 2014

  • Metzger, Meredith B.; Pruneda, Jonathan N.; Klevit, Rachel E.
  • Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol. 1843, Issue 1
  • DOI: 10.1016/j.bbamcr.2013.05.026

Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation
journal, August 2016


Ubiquitin and its binding domains
journal, January 2012

  • Randles, Leah
  • Frontiers in Bioscience, Vol. 17, Issue 7
  • DOI: 10.2741/4042

Lysine-targeting specificity in ubiquitin and ubiquitin-like modification pathways
journal, April 2014

  • Mattiroli, Francesca; Sixma, Titia K.
  • Nature Structural & Molecular Biology, Vol. 21, Issue 4
  • DOI: 10.1038/nsmb.2792

RING-Between-RING E3 Ligases: Emerging Themes amid the Variations
journal, November 2017


Priming and Extending: A UbcH5/Cdc34 E2 Handoff Mechanism for Polyubiquitination on a SCF Substrate
journal, March 2010


Posing the APC/C E3 Ubiquitin Ligase to Orchestrate Cell Division
journal, February 2019

  • Watson, Edmond R.; Brown, Nicholas G.; Peters, Jan-Michael
  • Trends in Cell Biology, Vol. 29, Issue 2
  • DOI: 10.1016/j.tcb.2018.09.007

A UbcH5/Ubiquitin Noncovalent Complex Is Required for Processive BRCA1-Directed Ubiquitination
journal, March 2006


Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C
journal, June 2016


The Ubiquitin Code
journal, July 2012


Principles of Ubiquitin-Dependent Signaling
journal, October 2018


Ubiquitin-Binding Proteins: Decoders of Ubiquitin-Mediated Cellular Functions
journal, July 2012


Noncovalent Ubiquitin Interactions Regulate the Catalytic Activity of Ubiquitin Writers
journal, November 2016

  • Wright, Joshua D.; Mace, Peter D.; Day, Catherine L.
  • Trends in Biochemical Sciences, Vol. 41, Issue 11
  • DOI: 10.1016/j.tibs.2016.08.003

RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex
journal, March 2015

  • Brown, Nicholas G.; VanderLinden, Ryan; Watson, Edmond R.
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 17
  • DOI: 10.1073/pnas.1504161112

Generation and Validation of Intracellular Ubiquitin Variant Inhibitors for USP7 and USP10
journal, November 2017

  • Zhang, Wei; Sartori, Maria A.; Makhnevych, Taras
  • Journal of Molecular Biology, Vol. 429, Issue 22
  • DOI: 10.1016/j.jmb.2017.05.025

Structural insights into non-covalent ubiquitin activation of the cIAP1-UbcH5B∼ubiquitin complex
journal, December 2018

  • Patel, Amrita; Sibbet, Gary J.; Huang, Danny T.
  • Journal of Biological Chemistry, Vol. 294, Issue 4
  • DOI: 10.1074/jbc.RA118.006045