To understand more about how the

To understand more about how the UbVs interact with USP15, structures of some of the UbV-USP15 complexes were solved. In one crystal structure, the DUSP-binding UbV forms a strand-swapped dimer whereby the first β strand of each monomer is flipped 180° to interact with its partner. The exchange creates a pocket, which embraces the DUSP domains of two USP15 molecules. This is not the first time that swapping of the first β strand in UbVs has been observed, suggesting that this kind of rearrangement is more commonly selected for than expected (Gabrielsen et al., 2017). Not surprisingly, the UbV-DUSP interface relies on the diversified residues of ubiquitin. On USP15 the interaction includes residues that are not conserved in USP4 and USP11, which probably accounts for the preference of DUSP-binding UbV for USP15. Teyra et al., (2019) also obtained structures of the UbVs from each successive library that bound to the catalytic domain of USP15. Surprisingly, none of the UbVs bind in the expected ubiquitin-binding pocket. Instead, all UbVs bind in a manner that is likely to occlude entry of ubiquitin into the active site of USP15 (Ward et al., 2018). While the UbV-USP15 interface is similar in all three of the structures, the interaction of the UbVs from libraries two and three is enhanced by an extended loop included as part of these advanced libraries. This loop makes extra contacts with USP15, but the contact site includes residues that are conserved with USP4, explaining why these UbVs increased non-specific binding. Together, the protein engineering approaches employed by Teyra et al., (2019) demonstrate the effectiveness of affinity maturation for improving the affinity of UbVs, but this comes at a cost in terms of specificity. Indeed, the most effective UbV was the dimeric construct generated from UbVs isolated from the initial library. Surprisingly, in lysophosphatidic acid sale to most reported UbVs, none of the USP15 UbVs bind in the known ubiquitin-binding sites. It is unclear if in USP15 the UbVs have selected cryptic ubiquitin or ubiquitin-like binding sites or whether the UbVs are more comparable to nanobodies in that they have selected a surface-exposed epitope that is unrelated to ubiquitin binding.
Acknowledgments We apologize to colleagues working in this field whose work was not cited due to space constraints. Our work is supported by grants from the Marsden Fund (NZ) and from the Health Research Council of New Zealand.
Introduction Proteins are vital for the structure and function of the cells, and the regulation of protein synthesis is a prime aspect of cellular metabolism. About 30% of mammalian proteins are short lived, have very short half-life of less than 10min and are rapidly degraded after translation (Schubert et al., 2000). Such a high level of protein degradation requires a dedicated system to regulate the selective unwanted protein degradation. Ubiquitin-proteasome system (UPS) has emerged as a key supervisor of protein function and stability. UPS has many vital roles in eukaryotic cellular processes including cell cycle progression, stress response, signal transduction, DNA repair, control of transcription factor activity and membrane trafficking (Coux et al., 1996, Hershko and Ciechanover, 1998, Ciechanover et al., 2000a, Ciechanover, 2006, Welchman et al., 2005). Ubiquitin plays an important role to degrade proteins through proteasome targeting as well as by direct sorting to the lysosome. Ubiquitin is a small eukaryotic polypeptide which marks unwanted or damaged proteins for degradation, and the proteasome, is a large molecule breaks down protein into smaller peptides, to be used in other anabolic processes (D\'Arcy et al., 2015). More than 80% of proteins are degraded by UPS and that is why it has emerged as an important player in the regulation of various cellular processes (Rock et al., 1994). UPS plays a pivotal role in the pathogenesis of many human diseases like cancer and neurodegenerative disorders (Ciechanover et al., 2000b).