Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

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.

PAICS ubiquitination recruits UBAP2 to trigger phase separation for purinosome assembly.

Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.

Structural basis of transcriptional activation by the OmpR/PhoB-family response regulator PmrA.

PmrA, an OmpR/PhoB-family response regulator, triggers gene transcription responsible for polymyxin resistance in bacteria by recognizing promoters where the canonical-35 element is replaced by the pmra-box, representing the PmrA recognition sequence. Here, we report a cryo-electron microscopy (cryo-EM) structure of a bacterial PmrA-dependent transcription activation complex (TAC) containing a PmrA dimer, an RNA polymerase σ70 holoenzyme (RNAPH) and the pbgP promoter DNA. Our structure reveals that the RNAPH mainly contacts the PmrA C-terminal DNA-binding domain (DBD) via electrostatic interactions and reorients the DBD three base pairs upstream of the pmra-box, resulting in a dynamic TAC conformation. In vivo assays show that the substitution of the DNA-recognition residue eliminated its transcriptional activity, while variants with altered RNAPH-interacting residues resulted in enhanced transcriptional activity. Our findings suggest that both PmrA recognition-induced DNA distortion and PmrA promoter escape play crucial roles in its transcriptional activation.

System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.

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.

Cryo-EM reveals the structure and dynamics of a 723-residue malate synthase G.

Determination of sub-100 kDa (kDa) structures by cryo-electron microscopy (EM) is a longstanding but not straightforward goal. Here, we present a 2.9-Å cryo-EM structure of a 723-amino acid apo-form malate synthase G (MSG) from Escherichia coli. The cryo-EM structure of the 82-kDa MSG exhibits the same global folding as structures resolved by crystallography and nuclear magnetic resonance (NMR) spectroscopy, and the crystal and cryo-EM structures are indistinguishable. Analyses of MSG dynamics reveal consistent conformational flexibilities among the three experimental approaches, most notably that the α/ß domain exhibits structural heterogeneity. We observed that sidechains of F453, L454, M629, and E630 residues involved in hosting the cofactor acetyl-CoA and substrate rotate differently between the cryo-EM apo-form and complex crystal structures. Our work demonstrates that the cryo-EM technique can be used to determine structures and conformational heterogeneity of sub-100 kDa biomolecules to a quality as high as that obtained from X-ray crystallography and NMR spectroscopy.

Cryo-EM analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans.

Feline infectious peritonitis virus (FIPV) is an alphacoronavirus that causes a nearly 100% mortality rate without effective treatment. Here we report a 3.3-Å cryoelectron microscopy (cryo-EM) structure of the serotype I FIPV spike (S) protein, which is responsible for host recognition and viral entry. Mass spectrometry provided site-specific compositions of densely distributed high-mannose and complex-type N-glycans that account for 1/4 of the total molecular mass; most of the N-glycans could be visualized by cryo-EM. Specifically, the N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV S protein result in a unique propeller-like conformation, underscoring the importance of glycosylation in maintaining protein structures. The cleavage site within the S2 subunit responsible for activation also showed distinct structural features and glycosylation. These structural insights provide a blueprint for a better molecular understanding of the pathogenesis of FIP.

2.2 Å Cryo-EM Tetra-Protofilament Structure of the Hamster Prion 108-144 Fibril Reveals an Ordered Water Channel in the Center.

Fibrils of the hamster prion peptide (sHaPrP, sequence 108-144) were prepared in an acidic solution, and their structure was solved by cryogenic electron microscopy with a resolution of 2.23 Å based on the gold-standard Fourier shell correlation (FSC) curve. The fibril has a novel architecture that has never been found in other amyloid fibrils. Each fibril is assembled by four protofilaments (PFs) and has an ordered water channel in the center. Each protofilament contains three ß-strands (125-130, 133-135, and 138-141) arranged in an "R"-shaped construct. The structural data indicate that these three ß-strand segments are the most amyloidogenic region of the prion peptide/protein and might be the site of nucleation during fibrillization under conditions without denaturants.

Direct Visualization of a 26 kDa Protein by Cryo-Electron Microscopy Aided by a Small Scaffold Protein.

Cryo-electron microscopy (cryo-EM)-based structure determination of small proteins is hindered by the technical challenges associated with low signal-to-noise ratios of their particle images in intrinsically noisy micrographs. One solution is to attach the target protein to a large protein scaffold to increase its apparent size and, therefore, image contrast. Here we report a novel scaffold design based on a trimeric helical protein, E. coli ornithine transcarbamylase (OTC), fused to human ubiquitin. As a proof of principle, we demonstrated the ability to resolve a cryo-EM map of a 26 kDa human ubiquitin C-terminal hydrolase (UCHL1) attached to the C-terminus of ubiquitin as part of the trimeric assembly. The results revealed conformational changes in UCHL1 upon binding to ubiquitin, namely, a significant displacement of α-helix 2, which was also observed by X-ray crystallography. Our findings demonstrated the potential of the trimeric OTC scaffold design for studying a large number of ubiquitin interacting proteins by cryo-EM.

Branched Ubiquitination: Detection Methods, Biological Functions and Chemical Synthesis.

Ubiquitination is a versatile posttranslational modification that elicits signaling roles to impact on various cellular processes and disease states. The versatility is a result of the complexity of ubiquitin conjugates, ranging from a single ubiquitin monomer to polymers with different length and linkage types. Recent studies have revealed the abundant existence of branched ubiquitin chains in which one ubiquitin molecule is connected to two or more ubiquitin moieties in the same ubiquitin polymer. Compared to the homotypic ubiquitin chain, the branched chain is recognized or processed differently by readers and erasers of the ubiquitin system, respectively, resulting in a qualitative or quantitative alteration of the functional output. Furthermore, certain types of branched ubiquitination are induced by cellular stresses, implicating their important physiological role in stress adaption. In addition, the current chemical methodologies of solid phase peptide synthesis and expanding genetic code approach have been developed to synthesize different architectures of branched ubiquitin chains. The synthesized branched ubiquitin chains have shown their significance in understanding the topologies and binding partners of the branched chains. Here, we discuss the recent progresses on the detection, functional characterization and synthesis of branched ubiquitin chains as well as the future perspectives of this emerging field.

A cascading activity-based probe sequentially targets E1-E2-E3 ubiquitin enzymes.

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like modifiers (Ubls), orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a wide range of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Similarly to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade, UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe 'hops' and 'traps' catalytically active Ub-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activity in living cells, presents novel and versatile tools to interrogate Ub and Ubl cascades.

Itch WW Domains Inhibit Its E3 Ubiquitin Ligase Activity by Blocking E2-E3 Ligase Trans-thiolation.

Nedd4-family E3 ubiquitin ligases regulate an array of biologic processes. Autoinhibition maintains these catalytic ligases in an inactive state through several mechanisms. However, although some Nedd4 family members are activated by binding to Nedd4 family-interacting proteins (Ndfips), how binding activates E3 function remains unclear. Our data reveal how these two regulatory processes are linked functionally. In the absence of Ndfip1, the Nedd4 family member Itch can bind an E2 but cannot accept ubiquitin onto its catalytic cysteine. This is because Itch is autoinhibited by an intramolecular interaction between its HECT (homologous to the E6-AP carboxy terminus domain) and two central WW domains. Ndfip1 binds these WW domains to release the HECT, allowing trans-thiolation and Itch catalytic activity. This molecular switch also regulates the closely related family member WWP2. Importantly, multiple PY motifs are required for Ndfip1 to activate Itch, functionally distinguishing Ndfips from single PY-containing substrates. These data establish a novel mechanism for control of the function of a subfamily of Nedd4 E3 ligases at the level of E2-E3 trans-thiolation.

ACA-specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase.

MazF is an mRNA interferase that cleaves mRNAs at a specific RNA sequence. MazF from E. coli (MazF-ec) cleaves RNA at A^CA. To date, a large number of MazF homologs that cleave RNA at specific three- to seven-base sequences have been identified from bacteria to archaea. MazF-ec forms a dimer, in which the interface between the two subunits is known to be the RNA substrate-binding site. Here, we investigated the role of the two loops in MazF-ec, which are closely associated with the interface of the MazF-ec dimer. We examined whether exchanging the loop regions of MazF-ec with those from other MazF homologs, such as MazF from Myxococcus xanthus (MazF-mx) and MazF from Mycobacterium tuberculosis (MazF-mt3), affects RNA cleavage specificity. We found that exchanging loop 2 of MazF-ec with loop 2 regions from either MazF-mx or MazF-mt3 created a new cleavage sequence at (A/U)(A/U)AA^C in addition to the original cleavage site, A^CA, whereas exchanging loop 1 did not alter cleavage specificity. Intriguingly, exchange of loop 2 with 8 or 12 consecutive Gly residues also resulted in a new RNA cleavage site at (A/U)(A/U)AA^C. The present study suggests a method for expanding the RNA cleavage repertoire of mRNA interferases, which is crucial for potential use in the regulation of specific gene expression and for biotechnological applications.

YeeU enhances the bundling of cytoskeletal polymers of MreB and FtsZ, antagonizing the CbtA (YeeV) toxicity in Escherichia coli.

All 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.

Fast hydrogen exchange affects ¹5N relaxation measurements in intrinsically disordered proteins.

Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, (1)H-(15)N HSQC, is used to measure the (15)N transverse relaxation rate (R2), the measured R2 rate is convoluted with the HX rate (kHX) and has higher apparent R2 values. Since the (15)N R2 measurement is important for analyzing protein backbone dynamics, the HX effect on the R2 measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing (15)N R 2 (CPMG) experiments on α-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R 2 (CPMG) can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D2O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R 2 (CPMG) values be obtained by methods described herein.

What Strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks.

Identifying critical residues in protein-protein binding and efficiently designing stable and specific protein binders to target another protein is challenging. In addition to direct contacts in a protein-protein binding interface, our study employs computation modeling to reveal the essential network of residue interaction and dihedral angle correlation critical in protein-protein recognition. We propose that mutating residues regions exhibited highly correlated motions within the interaction network can efficiently optimize protein-protein interactions to create tight and selective protein binders. We validated our strategy using ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complexes, where Ub is one central player in many cellular functions and PLpro is an antiviral drug target. Molecular dynamics simulations and experimental assays were used to predict and verify our designed Ub variant (UbV) binders. Our designed UbV with 3 mutated residues resulted in a ~3,500-fold increase in functional inhibition, compared with the wild-type Ub. Further optimization by incorporating 2 more residues within the network, the 5-point mutant achieved a KD of 1.5 nM and IC50 of 9.7 nM. The modification led to a 27,500-fold and 5,500-fold enhancements in affinity and potency, respectively, as well as improved selectivity, without destabilizing the UbV structure. Our study illustrates the importance of residue correlation and interaction networks in protein-protein interaction and introduces a new approach that can effectively design high affinity protein binder for cell biology studies and future therapeutic solution.

What Strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks.

Identifying critical residues in protein-protein binding and efficiently designing stable and specific protein binders is challenging. In addition to direct contacts in a protein-protein binding interface, our study employs computation modeling to reveal the essential network of residue interaction and dihedral angle correlation critical in protein-protein recognition. We propose that mutating residues regions exhibited highly correlated motions within the interaction network can efficiently optimize protein-protein interactions to create tight and selective protein binders. We validated our strategy using ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complexes, where Ub is one central player in many cellular functions and PLpro is an antiviral drug target. Our designed UbV with 3 mutated residues resulted in a ~3,500-fold increase in functional inhibition, compared with the wild-type Ub. Further optimization by incorporating 2 more residues within the network, the 5-point mutant achieved a KD of 1.5 nM and IC50 of 9.7 nM. The modification led to a 27,500-fold and 5,500-fold enhancements in affinity and potency, respectively, as well as improved selectivity, without destabilizing the UbV structure. Our study highlights residue correlation and interaction networks in protein-protein interaction, introduces an effective approach to design high affinity protein binders for cell biology and future therapeutics solutions.

What Strengthens Protein-Protein Interactions: Analysis and Applications of Residue Correlation Networks.

Identifying residues critical to protein-protein binding and efficient design of stable and specific protein binders are challenging tasks. Extending beyond the direct contacts in a protein-protein binding interface, our study employs computational modeling to reveal the essential network of residue interactions and dihedral angle correlations critical in protein-protein recognition. We hypothesized that mutating residues exhibiting highly correlated dynamic motion within the interaction network could efficiently optimize protein-protein interactions to create tight and selective protein binders. We tested this hypothesis using the ubiquitin (Ub) and MERS coronaviral papain-like protease (PLpro) complex, since Ub is a central player in multiple cellular functions and PLpro is an antiviral drug target. Our designed ubiquitin variant (UbV) hosting three mutated residues displayed a ∼3,500-fold increase in functional inhibition relative to wild-type Ub. Further optimization of two C-terminal residues within the Ub network resulted in a KD of 1.5 nM and IC50 of 9.7 nM for the five-point Ub mutant, eliciting 27,500-fold and 5,500-fold enhancements in affinity and potency, respectively, as well as improved selectivity, without destabilizing the UbV structure. Our study highlights residue correlation and interaction networks in protein-protein interactions, and introduces an effective approach to design high-affinity protein binders for cell biology research and future therapeutics.

Robust Design of Effective Allosteric Activators for Rsp5 E3 Ligase Using the Machine Learning Tool ProteinMPNN.

We used the deep learning tool ProteinMPNN to redesign ubiquitin (Ub) as a specific and functionally stimulating/enhancing binder of the Rsp5 E3 ligase. We generated 20 extensively mutated─up to 37 of 76 residues─recombinant Ub variants (UbVs), named R1 to R20, displaying well-folded structures and high thermal stabilities. These UbVs can also form stable complexes with Rsp5, as predicted using AlphaFold2. Three of the UbVs bound to Rsp5 with low micromolar affinity, with R4 and R12 effectively enhancing the Rsp5 activity six folds. AlphaFold2 predicts that R4 and R12 bind to Rsp5's exosite in an identical manner to the Rsp5-Ub template, thereby allosterically activating Rsp5-Ub thioester formation. Thus, we present a virtual solution for rapidly and cost-effectively designing UbVs as functional modulators of Ub-related enzymes.

TRABID inhibition activates cGAS/STING-mediated anti-tumor immunity through mitosis and autophagy dysregulation.

Activation of tumor-intrinsic innate immunity has been a major strategy for improving immunotherapy. Previously, we reported an autophagy-promoting function of the deubiquitinating enzyme TRABID. Here, we identify a critical role of TRABID in suppressing anti-tumor immunity. Mechanistically, TRABID is upregulated in mitosis and governs mitotic cell division by removing K29-linked polyubiquitin chain from Aurora B and Survivin, thereby stabilizing the entire chromosomal passenger complex. TRABID inhibition causes micronuclei through a combinatory defect in mitosis and autophagy and protects cGAS from autophagic degradation, thereby activating the cGAS/STING innate immunity pathway. Genetic or pharmacological inhibition of TRABID promotes anti-tumor immune surveillance and sensitizes tumors to anti-PD-1 therapy in preclinical cancer models in male mice. Clinically, TRABID expression in most solid cancer types correlates inversely with an interferon signature and infiltration of anti-tumor immune cells. Our study identifies a suppressive role of tumor-intrinsic TRABID in anti-tumor immunity and highlights TRABID as a promising target for sensitizing solid tumors to immunotherapy.

PTPN23 ubiquitination by WDR4 suppresses EGFR and c-MET degradation to define a lung cancer therapeutic target.

Aberrant overexpression or activation of EGFR drives the development of non-small cell lung cancer (NSCLC) and acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) by secondary EGFR mutations or c-MET amplification/activation remains as a major hurdle for NSCLC treatment. We previously identified WDR4 as a substrate adaptor of Cullin 4 ubiquitin ligase and an association of WDR4 high expression with poor prognosis of lung cancer. Here, using an unbiased ubiquitylome analysis, we uncover PTPN23, a component of the ESCRT complex, as a substrate of WDR4-based ubiquitin ligase. WDR4-mediated PTPN23 ubiquitination leads to its proteasomal degradation, thereby suppressing lysosome trafficking and degradation of wild type EGFR, EGFR mutant, and c-MET. Through this mechanism, WDR4 sustains EGFR and c-MET signaling to promote NSCLC proliferation, migration, invasion, stemness, and metastasis. Clinically, PTPN23 is downregulated in lung cancer and its low expression correlates with WDR4 high expression and poor prognosis. Targeting WDR4-mediated PTPN23 ubiquitination by a peptide that competes with PTPN23 for binding WDR4 promotes EGFR and c-MET degradation to block the growth and progression of EGFR TKI-resistant NSCLC. These findings identify a central role of WDR4/PTPN23 axis in EGFR and c-MET trafficking and a potential therapeutic target for treating EGFR TKI-resistant NSCLC.