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

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.

doi:10.1073/pnas.1902889116

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

Journal Article · Fri Jul 26 00:00:00 EDT 2019 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1902889116· OSTI ID:1562701

Watson, Edmond R. ^[1]; Grace, Christy R. R. ^[2]; Zhang, Wei ^[3]; Miller, Darcie J. ^[2]; Davidson, Iain F. ^[4]; Prabu, J. Rajan ^[5]; Yu, Shanshan ^[2]; Bolhuis, Derek L. ^[6]; Kulko, Elizaveta T. ^[6]; Vollrath, Ronnald ^[5]; Haselbach, David ^[7]; Stark, Holger ^[8]; Peters, Jan-Michael ^[9]; Brown, Nicholas G. ^[10]; Sidhu, Sachdev S. ^[3]; Schulman, Brenda A. ^[1]

St. Jude Children’s Research Hospital, Memphis, TN (United States); Max Planck Inst. of Biochemistry, Martinsried (Germany)
St. Jude Children’s Research Hospital, Memphis, TN (United States)
Univ. of Toronto, ON (Canada)
Vienna BioCenter (Austria)
Max Planck Inst. of Biochemistry, Martinsried (Germany)
Univ. of North Carolina, Chapel Hill, NC (United States). Lineberger Comprehensive Cancer Center
Vienna BioCenter (Austria); Max Planck Inst. for Biophysical Chemistry, Göttingen (Germany)
Max Planck Inst. for Biophysical Chemistry, Göttingen (Germany)
Vienna BioCenter (Austria); Medical Univ. of Vienna (Austria)
St. Jude Children’s Research Hospital, Memphis, TN (United States); Univ. of North Carolina, Chapel Hill, NC (United States). Lineberger Comprehensive Cancer Center

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.

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Research Organization:: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: USDOE; German Research Foundation (DFG); Austrian Research Promotion Agency; European Union (EU); Austrian Science Fund (FWF); National Institutes of Health (NIH); Genome Canada

Grant/Contract Number:: 860; 227764; SFB-F34; R35GM128855; R37GM065930; P30CA021765; OGI-119

OSTI ID:: 1562701

Journal Information:: Proceedings of the National Academy of Sciences of the United States of America, Vol. 116, Issue 35; ISSN 0027-8424

Publisher:: National Academy of SciencesCopyright Statement

Country of Publication:: United States

Language:: ENGLISH

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Cited by: 15 works

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

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