Summary In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2~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 Cys 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~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.

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

Post-translational covalent modification by ubiquitin and ubiquitin-like proteins (UBLs) is a major eukaryotic mechanism for regulating protein function. In general, each UBL has its own E1 that serves as the entry point for a cascade. The E1 first binds the UBL and catalyzes adenylation of the UBL’s C-terminus, prior to promoting UBL transfer to a downstream E2. Ubiquitin’s Arg 72, which corresponds to Ala72 in the UBL NEDD8, is a key E1 selectivity determinant: swapping ubiquitin and NEDD8 residue 72 identity was shown previously to swap their E1 specificity. Correspondingly, Arg190 in the UBA3 subunit of NEDD8’s heterodimeric E1 (the APPBP1-UBA3 complex), which corresponds to a Gln in ubiquitin’s E1 UBA1, is a key UBL selectivity determinant. Here, we dissect this specificity with biochemical and X-ray crystallographic analysis of APPBP1-UBA3-NEDD8 complexes in which NEDD8’s residue 72 and UBA3’s residue 190 are substituted with different combinations of Ala, Arg, or Gln. APPBP1-UBA3’s preference for NEDD8’s Ala72 appears to be indirect, due to proper positioning of UBA3’s Arg190. By contrast, our data are consistent with direct positive interactions between ubiquitin’s Arg72 and an E1’s Gln. However, APPBP1-UBA3’s failure to interact with a UBL having Arg72 is not due to a lack of this favorable interaction, but rather arises from UBA3’s Arg190 acting as a negative gate. Thus, parallel residues from different UBL pathways can utilize distinct mechanisms to dictate interaction selectivity, and specificity can be amplified by barriers that prevent binding to components of different conjugation cascades.

A protocol for purifying tubulin with controlled posttranslational modifications using cycles of polymerisation and depolymerisation. Tubulin can be obtained from cultured cells or single mouse brains and used for in vitro reconstitution assays. TWEET A new protocol for purifying tubulin with controlled posttranslational modifications for in vitro reconstitution experiments.

Despite the availability of antibiotics and vaccines, Neisseria meningitidis remains a major cause of meningitis and sepsis in humans. Due to its extracellular lifestyle, bacterial adhesion to host cells constitutes an attractive therapeutic target. Here, we present a high-throughput microscopy-based approach that allowed the identification of compounds able to decrease type IV pilus-mediated interaction of bacteria with endothelial cells in the absence of bacterial or host cell toxicity. Compounds specifically inhibit the PilF ATPase enzymatic activity that powers type IV pilus extension but remain inefficient on the ATPase that promotes pilus retraction, thus leading to rapid pilus disappearance from the bacterial surface and loss of pili-mediated functions. Structure activity relationship of the most active compound identifies specific moieties required for the activity of this compound and highlights its specificity. This study therefore provides compounds targeting pilus biogenesis, thereby inhibiting bacterial adhesion, and paves the way for a novel therapeutic option for meningococcal infections. meningitis | bacteria | virulence | inhibitors | type IV pilus

Posttranslational modifications of tubulin currently emerge as key regulators of microtubule functions. Polyglutamylation generates a variety of modification patterns that are essential for controlling microtubule functions in different cell types and organelles, and deregulation of these patterns has been linked to ciliopathies, cancer and neurodegeneration. How the different glutamylating enzymes determine precise modification patterns has so far remained elusive. Using computational modelling, molecular dynamics simulations and mutational analyses we now show how the carboxy-terminal tails of tubulin bind into the active sites of glutamylases. Our models suggest that the glutamylation sites on α- and β-tubulins are determined by the positioning of the tails within the catalytic pocket. Moreover, we found that the binding modes of α- and β-tubulin tails are highly similar, implying that most enzymes could potentially modify both, α- and β-tubulin. This supports a model in which the binding of the enzymes to the entire microtubule lattice, but not the specificity of the C-terminal tubulin tails to their active sites, determines the catalytic specificities of glutamylases.

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB, binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB (RI-MUB) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB-Cy5 using live cell microscopy. Systemically administered RI-MUB-Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB marker will aid the study of broad ranges of inflammatory diseases.

Most lncRNAs display species-specific expression patterns suggesting that animal models of cancer may only incompletely recapitulate the regulatory crosstalk between lncRNAs and oncogenic pathways in humans. Among these pathways, Sonic Hedgehog (SHH) signaling is aberrantly activated in several human cancer entities. We unravel that aberrant expression of the primate-specific lncRNA HedgeHog Interacting Protein-AntiSense 1 (HHIP-AS1) is a hallmark of SHH-driven tumors including medulloblastoma and atypical teratoid/rhabdoid tumors. HHIP-AS1 is actively transcribed from a bidirectional promoter shared with SHH regulator HHIP. Knockdown of HHIP-AS1 induces mitotic spindle deregulation impairing tumorigenicity in vitro and in vivo. Mechanistically, HHIP-AS1 binds directly to the mRNA of cytoplasmic dynein 1 intermediate chain 2 (DYNC1I2) and attenuates its degradation by hsa-miR-425-5p. We uncover that neither HHIP-AS1 nor the corresponding regulatory element in DYNC1I2 are evolutionary conserved in mice. Taken together, we discover an lncRNA-mediated mechanism that enables the pro-mitotic effects of SHH pathway activation in human tumors.