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.

Yeast reveal a "druggable" Rsp5/Nedd4 network that ameliorates α-synuclein toxicity in neurons.

α-Synuclein (α-syn) is a small lipid-binding protein implicated in several neurodegenerative diseases, including Parkinson's disease, whose pathobiology is conserved from yeast to man. There are no therapies targeting these underlying cellular pathologies, or indeed those of any major neurodegenerative disease. Using unbiased phenotypic screens as an alternative to target-based approaches, we discovered an N-aryl benzimidazole (NAB) that strongly and selectively protected diverse cell types from α-syn toxicity. Three chemical genetic screens in wild-type yeast cells established that NAB promoted endosomal transport events dependent on the E3 ubiquitin ligase Rsp5/Nedd4. These same steps were perturbed by α-syn itself. Thus, NAB identifies a druggable node in the biology of α-syn that can correct multiple aspects of its underlying pathology, including dysfunctional endosomal and endoplasmic reticulum-to-Golgi vesicle trafficking.

Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3.

Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI:http://dx.doi.org/10.7554/eLife.00828.001.

Insights into ubiquitin transfer cascades from a structure of a UbcH5B approximately ubiquitin-HECT(NEDD4L) complex.

In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2 approximately Ub covalent complex and then interacts with an E3 for Ub transfer. For cascades involving E3s in the HECT class, Ub is transferred from an associated E2 to the acceptor cysteine in the HECT domain C lobe. To gain insights into this process, we determined the crystal structure of a complex between the HECT domain of NEDD4L and the E2 UbcH5B bearing a covalently linked Ub at its active site (UbcH5B approximately Ub). Noncovalent interactions between UbcH5B and the HECT N lobe and between Ub and the HECT domain C lobe lead to an overall compact structure, with the Ub C terminus sandwiched between UbcH5B and HECT domain active sites. The structure suggests a model for E2-to-HECT Ub transfer, in which interactions between a donor Ub and an acceptor domain constrain upstream and downstream enzymes for conjugation.

Crystallization and structure determination of the core-binding domain of bacteriophage lambda integrase.

Bacteriophage lambda integrase catalyzes site-specific DNA recombination. A helical bundle domain in the enzyme, called the core-binding domain (Int(CB)), promotes the catalysis of an intermediate DNA-cleavage reaction that is critical for recombination and is not well folded in solution in the absence of DNA. To gain structural insights into the mechanism behind the accessory role of this domain in catalysis, an attempt was made to crystallize an Int(CB)-DNA complex, but crystals of free Int(CB) were fortuitously obtained. The three-dimensional structure of DNA-free Int(CB) was solved at 2.0 A resolution by molecular replacement using as the search model the previously available DNA-bound 2.8 A structure of the Int(CB) domain in a larger construct of lambda integrase. The crystal structure of DNA-free Int(CB) resembles the DNA-bound structure of Int(CB), but exhibits subtle differences in the DNA-binding face and lacks electron density for ten residues in the C-terminus that form a portion of a linker connecting Int(CB) to the C-terminal catalytic domain of the enzyme. Thus, this work reveals the domain in the absence of DNA and allows comparison with the DNA-bound form of this catalytically activating domain.

DNA recognition via mutual-induced fit by the core-binding domain of bacteriophage lambda integrase.

Bacteriophage lambda integrase (lambda-Int), a phage-encoded DNA recombinase, cleaves its substrate DNA to facilitate the formation and later resolution of a Holliday junction intermediate during recombination. The core-binding and catalytic domains of lambda-Int constitute a bipartite enzyme that mediates site-specific DNA cleavage through their interactions with opposite sides of the recognition sequence. Despite minimal direct contact between the domains, the core-binding domain has been shown to facilitate site-specific DNA cleavage when provided in trans, indicating that it plays a role beyond enhancing binding affinity. Biophysical characterization of the core-binding domain and its interactions with DNA reveal that the domain is poorly structured in its free form and folds upon binding to DNA. Folding of the protein is accompanied by induced-fit structural changes in the DNA ligand. These data support a model by which the core-binding domain plays a catalytic role by reshaping the substrate DNA for effective cleavage by the catalytic domain.

Trans cooperativity by a split DNA recombinase: the central and catalytic domains of bacteriophage lambda integrase cooperate in cleaving DNA substrates when the two domains are not covalently linked.

Site-specific recombinases of the lambda-integrase family recognize and cleave their cognate DNA sites through cooperative binding to opposite sides of the DNA substrate by a C-terminal catalytic domain and a flexibly linked "core-binding" domain; regulation of this cleavage is achieved via the formation of higher-order complexes. We report that the core-binding domain of lambda-integrase is able to stimulate the activity of the catalytic domain even when the two domains are not linked. This trans stimulation is accomplished without significantly increasing the affinity of the catalytic domain for its DNA substrate. Moreover, we show that mutations in the DNA substrate can abrogate this effect while retaining specificity determinants for cleavage. Since the domains do not significantly interact directly, this finding implies that trans activation is achieved via the DNA substrate in a manner that may be mechanistically important in this and similar DNA binding and cleaving enzymes.

Protein folding coupled to DNA binding in the catalytic domain of bacteriophage lambda integrase detected by mass spectrometry.

Bacteriophage lambda integrase (lambda-Int) is the prototypical member of a large family of enzymes that catalyze site-specific DNA recombination via single-strand cleavage and the formation of a Holliday junction intermediate. Crystallographic and biochemical evidence indicate that substantial conformational change (i.e., folding) in the catalytic domain of the protein is required for substrate recognition and catalysis. We have examined the solution conformation of the catalytic domain (C170) in the absence and presence of a cognate "half-site" DNA oligonucleotide by electrospray ionization mass spectrometry, and circular dichroism and fluorescence spectroscopy. The distribution of ions in the positive ion electrospray mass spectrum of the free protein reveals the presence of three distinct species in solution, one corresponding to the folded protein, one to the unfolded protein, and one to a dimer. In the presence of DNA, ions are observed only for the protein-DNA complex and the folded form of the free protein. We therefore conclude that DNA binding stabilizes the global fold of the protein in a manner that is consistent with folding-coupled target recognition as a mechanism to control site-specific recombination. Furthermore, we find that inspection of the charge state distribution of ions in electrospray mass spectra provides a quick and effective means to identify conformational heterogeneity of proteins in solution and to investigate dynamic protein-nucleic acid interactions.