SUMMARY HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process, and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site, and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.

SUMMARY Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3–E2–substrate-UB architectures control different forms of polyubiquitination.

Summaryα-synuclein is an intrinsically disordered protein that appears in aggregated forms in the brains of patients with Parkinson's Disease. The conversion from monomer to aggregate is complex and aggregation rates are sensitive to changes in amino acid sequence and environmental conditions. It has previously been observed that α-synuclein aggregates faster at low pH than at neutral pH. Here, we combine NMR spectroscopy and molecular simulations to characterize α-synuclein conformational ensembles at both neutral and low pH in order to understand how the altered charge distribution at low pH changes the structural properties of these ensembles and leads to an increase in aggregation rate. The N-terminus, which has a small positive charge at neutral pH due to a balance of positively and negatively charged amino acid residues, is very positively charged at low pH. Conversely, the acidic C-terminus is highly negatively charged at neutral pH and becomes essentially neutral and hydrophobic at low pH. Our NMR experiments and REMD simulations indicate that there is a significant structural reorganization within the low pH ensemble relative to that at neutral pH in terms of long range contacts, hydrodynamic radius, and the amount of heterogeneity within the conformational ensembles. At neutral pH there is a very heterogeneous ensemble with transient contacts between the N-terminus and the NAC, however at low pH there is a more homogeneous ensemble which exhibits strong contacts between the NAC and the C-terminus. At both pHs, transient contacts between the N-and C-termini are observed, the NAC region shows similar exposure to solvent, and the entire protein shows similar propensities to secondary structure. Based on the comparison of the neutral and low pH conformational ensembles, we propose that exposure of the NAC region to solvent and the secondary structure propensity are not factors that account for differences in propensity to aggregate in this context. Instead, the comparison of the neutral and low pH ensembles suggests that the change in long-range interactions between the low and neutral pH ensembles, the compaction of the C-terminal region at low pH and the uneven distribution of charges across the sequence are key to faster aggregation.

SummaryAll free-living bacteria carry the toxin-antitoxin (TA) systems controlling cell growth and death under stress conditions. YeeU-YeeV (CbtA) is one of the Escherichia coli TA systems, and the toxin, CbtA, has been reported to inhibit the polymerization of bacterial cytoskeletal proteins, MreB and FtsZ. Here, we demonstrate that the antitoxin, YeeU, is a novel type of antitoxin (type IV TA system), which does not form a complex with CbtA but functions as an antagonist for CbtA toxicity. Specifically, YeeU was found to directly interact with MreB and FtsZ, and enhance the bundling of their filamentous polymers in vitro. Surprisingly, YeeU neutralized not only the toxicity of CbtA but also the toxicity caused by other inhibitors of MreB and FtsZ, such as A22, SulA and MinC, indicating that YeeU-induced bundling of MreB and FtsZ has an intrinsic global stabilizing effect on their homeostasis. Here we propose to rename YeeU as CbeA for cytoskeleton bundling-enhancing factor A.

SummaryConversion of human α-synuclein (aS) from the free soluble state to the insoluble fibrillar state has been implicated in the etiology of Parkinson's disease. Human aS is highly homologous in amino acid sequence to mouse aS which contains seven substitutions including the A53T substitution that is the same as the one that has been linked to familial Parkinson's disease, and including five substitutions in the C terminal region. It has been shown that the rate of fibrillation is highly dependent on the exact sequence of the protein and mouse aS is reported to aggregate more rapidly than human aS in vitro. Nuclear magnetic resonance of mouse aS and human aS at supercooled temperatures (263K) is used to understand the effect of sequence on conformational fluctuations in the disordered ensembles and to relate these to differences in propensities to aggregate. We show that human and mouse aS are natively unfolded at low temperature but that they exhibit different propensities to secondary structure, backbone dynamics and long range contacts across the protein.Mouse aS exhibits a higher propensity to helical conformation at the C terminal substitution sites as well as the loss of transient long range contacts from the C terminal end to the N terminal and hydrophobic central region of the protein relative to human aS. Lack of back-folding from the C terminal end of mouse aS exposes the N terminal region which is shown, by 15 N relaxation experiments, to be very restricted in mobility relative to human aS. We propose that the restricted mobility in the N terminal region may arise from transient interchain interactions suggesting that the N-terminal KTK(E/Q)GV hexamer repeats may serve as initiation sites for aggregation in mouse aS. These transient interchain interactions of the N terminal region coupled with a non-Aβ amyloid component (NAC) region that is both more exposed and has a higher propensity to β structure may explain the increased rate of fibril formation of mouse aS relative to human aS and A53T aS.