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

Watson, E. R., Grace, C. R. R., Zhang, W., Miller, D. J., Davidson, I. F., Prabu, J. R., et al. (2019). Protein engineering of a ubiquitin-variant inhibitor of APC/C identifies a cryptic K48 ubiquitin chain binding site. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 116(35), 17280-17289. doi:10.1073/pnas.1902889116.

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 Creators:
Watson, Edmond R.1, Author           
Grace, Christy R. R.2, Author
Zhang, Wei2, Author
Miller, Darcie J.2, Author
Davidson, Iain F.2, Author
Prabu, J. Rajan1, Author           
Yu, Shanshan2, Author
Bolhuis, Derek L.2, Author
Kulko, Elizaveta T.2, Author
Vollrath, Ronnald1, Author           
Haselbach, David2, Author
Stark, Holger2, Author
Peters, Jan-Michael2, Author
Brown, Nicholas G.2, Author
Sidhu, Sachdev S.2, Author
Schulman, Brenda A.1, Author           
Affiliations:
1Schulman, Brenda / Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Max Planck Society, ou_2466699              
2external, ou_persistent22              

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Free keywords: ANAPHASE-PROMOTING COMPLEX; E3 LIGASES; FUNCTIONAL-CHARACTERIZATION; MECHANISM; RING; ELONGATION; CATALYSIS; INSIGHTS; E2; POLYUBIQUITINATIONScience & Technology - Other Topics; anaphase-promoting complex; APC/C; ubiquitin; E3 ligase; inhibitor;
 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. Here, 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.

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Language(s): eng - English
 Dates: 2019
 Publication Status: Published in print
 Pages: 10
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: ISI: 000483396800027
DOI: 10.1073/pnas.1902889116
 Degree: -

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Title: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Source Genre: Journal
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Publ. Info: 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA : NATL ACAD SCIENCES
Pages: - Volume / Issue: 116 (35) Sequence Number: - Start / End Page: 17280 - 17289 Identifier: ISSN: 0027-8424