Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 sidechain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2~Ub conjugates.

S100A6 is a member of the S100 subfamily of Ca2+ binding EF-hand proteins that has been shown to interact with calcyclin binding protein/Siah-1 interacting protein (CacyBP/SIP; SIP), a subunit of an SCF-like E3 ligase complex (SCF-TBL1) formed under genotoxic stress. SIP serves as a scaffold in this complex, linking the E2-recruiting module Siah-1 to the substrate-recruiting module Skp1-TBL1. A cell-based functional assay suggests that S100A6 modulates the activity of SCF-TBL1. The results from the cell-based experiments could be enhanced if it were possible to selectively inhibit S100A6-SIP interactions without perturbing any other functions of the two proteins. To this end, the structure of the S100A6-SIP complex was determined in solution by NMR and the strength of the interaction was characterized by isothermal titration calorimetry. In an initial step, the minimal binding region in SIP for S100A6 was mapped to a 31 residue fragment (Ser189-Arg219) in the C-terminal domain. The structure of the S100A6-SIP(189–219) complex revealed that SIP(189–219) forms two helices, the first of which (Met193-Tyr200) interacts with S100A6 in a canonical binding mode. The second helix (Met207-Val216) lies over the S100A6 dimer interface, a mode of binding to S100A6 that has not previously been observed for any target bound to an S100 protein. A series of structure-based SIP mutations showed reduced S100A6 binding affinity, setting the stage for direct functional analysis of S100A6-SIP interactions.

Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1. In this report, we show that alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. We also identify a novel ubiquitin-interacting motif (UIM) within alpha4 (amino acid residues [46][47][48][49][50][51][52][53][54][55][56][57][58][59][60] and analyze the interaction between alpha4 and ubiquitin using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, we demonstrate that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4.Protein serine/threonine phosphatase 2A (PP2A) is an abundant cellular enzyme with numerous substrates that modulate a wide variety of cellular functions. Considering the multitude of cellular processes under the control of PP2A, it is not surprising that several different mechanisms exist to regulate phosphatase activity. These regulatory mechanisms include association with specific regulatory subunits and post-translational modifications of PP2Ac (i.e. phosphorylation, carboxymethylation, and ubiquitination) (1-3). Both biochemical and structural studies of PP2A have provided key mechanistic insights to explicate regulation of phosphatase holoenzyme composition and activity via phosphorylation and carboxymethylation (1,2,4,5); however, little is known about PP2Ac ubiquitination beyond the initial report demonstrating the polyubiquitination and degradation of microtubule-associated PP2Ac (3). The E3 ubiquitin ligase responsible for targeting PP2Ac for proteasome degradation