br Results br Discussion Our UbV library was originally

Results
Discussion Our UbV library was originally designed to develop inhibitors of deubiquitinases (Ernst et al., 2013). Recently, we showed that UbVs could exhibit multiple binding modes and mechanisms to modulate HECT E3 activity (Zhang et al., 2016)—one set occupied the HECT domain E2-binding site and inhibited Ub ligation. Here, our structural studies show that the E2∼Ub-binding site of RING/U-box is also targetable by UbVs. Therefore, a generalizable strategy is now available to potentially generate potent and specific inhibitors for over 300 human RING/U-box domains. Our study of HECT E3s also identified UbVs that bind to a Ub-binding exosite and stimulate ubiquitination activity (Zhang et al., 2016). In Pam3CSK4 Biotin to HECT domains, in which a large surface area of ∼19,000 Å2 is available for binding, RING and U-box domains only present a total surface area of ∼5,000 Å2 or ∼7,600 Å2 as monomers or dimers, respectively. Given this relatively small surface area, it is noteworthy that UbV.XR activates E3 activity through binding to a region on XIAP that has not previously been reported to be involved in protein-protein interactions. These data demonstrate the versatility of our UbV technology for exploiting both canonical and non-canonical protein-protein interaction surfaces of RING and U-box domains to develop selective probes. All three UbVs were active in vitro and in cells, thereby providing valuable tools for probing the cellular functions of UBE4B, CBL, and XIAP. In addition to regulating p53 stability and function (Wu et al., 2011), ubiquitination by UBE4B also plays a role in endosomal sorting and lysosomal degradation of EGFR and probably regulates sorting of other membrane proteins (Sirisaengtaksin et al., 2014). UbV.E4B will be useful for discovering additional pathways subject to UBE4B-mediated ubiquitination. CBL functions as both an adaptor and a negative regulator in tyrosine kinase-mediated signaling. It attenuates signaling by ubiquitinating receptor and non-receptor tyrosine kinases and targeting them for degradation. Substrate ubiquitination involves the N-terminal region of CBL encompassing the TKBD, linker, and RING domain and requires phosphorylation of Tyr371, whereas the adaptor functions of CBL are mediated by a proline-rich region and the C terminus (Levkowitz et al., 1999, Swaminathan and Tsygankov, 2006). UbV.pCBL only binds CBL when it is phosphorylated on Tyr371, thus providing a tool to differentiate between its ligase and adaptor functions in cells. XIAP inhibits apoptosis by binding Caspase-3, -7, and -9, and it activates NFκB signaling by binding the upstream adaptor TAB1. Moreover, it is upregulated in several cancers and confers resistance to chemotherapy-induced cell death (Obexer and Ausserlechner, 2014). Small molecule inhibitors, such as SMAC mimetics, have been developed to target the baculovirus-IAP-repeat (BIR) domain, but these molecules also target the BIR domains of the inhibitor of apoptosis family E3s (Bai et al., 2014). The E3 ligase activity of the RING domain of XIAP controls its stability and ubiquitinates substrates such as Caspase-3 and mSMAC (MacFarlane et al., 2002, Suzuki et al., 2001). The specificity of UbV.XR for XIAP and its role as a stimulator of E3 activity will enable further studies of hyperactive XIAP in cells and will enhance understanding of how E3 activity could influence its function. Our data suggest that UbV.XR stimulates XIAP-mediated Ub transfer by binding the RING domain and stabilizing donor Ub in a conformation primed for catalysis. Only one other RING E3 has a similar mechanism: free Ub binds the RING domain of the monomeric E3 Arkadia and is postulated to directly contact and position donor Ub for transfer (Wright et al., 2016). However, UbV.XR and free Ub bind to different regions of the XIAP and Arkadia RING domains, respectively, and consequently contact different surfaces on donor Ub. Moreover, our data indicate that UbV.XR must dimerize to stimulate XIAP-mediated Ub transfer.