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. 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.

Ubiquitin in disguise unveils a cryptic binding site in 1.2-MDa anaphase-promoting complex/cyclosome

“…A crystal structure of UbV W revealed a domain-swapped dimer with an extended β1 strand that forms an antiparallel β-sheet with the opposite subunit (6). Watson et al (6) note that such swapped dimeric structures have been observed in other UbVs (14,15) and collectively display flexibility across the dimer interface. In UbV W , Gly10 is replaced with tryptophan and situated at the center of the extended β1 strand (6).…”

“…Identifying receptor sites for ubiquitin de novo is challenging, as ubiquitin-binding domains are diverse in structure and binding mechanism, resulting in no globally shared consensus sequence (5). In PNAS, Watson et al (6) present a workflow that can be applied broadly to discover new ubiquitin-binding sites. They apply this method to the 1.2-MDa E3 ligase anaphase-promoting complex/cyclosome (APC/C), which promotes ubiquitindependent turnover of cell cycle regulators, including cyclins, to control cell division.…”

“…In a previous study, phage-displayed libraries were used to generate ubiquitin variants (UbVs) that target the APC11 RING domain for inhibition of multiubiquitination by UBE2C and ubiquitin chain elongation by UBE2S (11). In a new study, Watson et al (6) identify a UbV that selectively inhibits UBE2C and not UBE2S activity. By combining protein engineering, biochemistry assays, and NMR spectroscopy, Watson et al (6) find this UbV inhibitor of APC/C to block UBE2C substrate priming by occupying the UBE2C-binding site in the APC2 WHB domain and hence name it UbV W .…”

“…In a new study, Watson et al (6) identify a UbV that selectively inhibits UBE2C and not UBE2S activity. By combining protein engineering, biochemistry assays, and NMR spectroscopy, Watson et al (6) find this UbV inhibitor of APC/C to block UBE2C substrate priming by occupying the UBE2C-binding site in the APC2 WHB domain and hence name it UbV W . The UBE2C-binding site in APC2 is remote from where the elongating E2 UBE2S binds (9) and is apparently disposable for UBE2S-catalyzed polyubiquitination, as UbV W did not affect this activity.…”

“…In UbV W , Gly10 is replaced with tryptophan and situated at the center of the extended β1 strand (6). By using amino acid substitution, Watson et al (6) find this tryptophan to be critical for oligomerization and to play a role in APC2 binding.…”