Characterisation of the deubiquitinating enzyme USP20

USP20 is a deubiquitinating enzyme that is involved in a number of important cellular pathways, including thyroid metabolism, hypoxic response, seven transmembrane receptor signalling, NF-κβ signalling, centrosome homeostasis and DNA repair. Of recent, it is becoming a major deubiquitinase involved in regulating the DNA-damage response pathway and cell cycle checkpoints. The protein consists of a zinc finger domain, catalytic domain and two ‘domain present in USP’ (DUSP) domains; an architecture shared only with its paralogue USP33. There is no structural information on any of the domains of USP20, so crystallisation trials of the domains of USP20 were performed in order to solve their structures by X-ray crystallography. In addition, yeast two-hybrid (Y2H) and in vitro assays were used to further characterise known and putative interactors of USP20. Finally, the zinc finger domain and DUSP domains were used in pull down assays to identify USP20-interacting proteins from HEK293 lysate. Two stable and well-expressing constructs of the zing finger domain (USP20 1-101 and 1-108) were purified and set up for crystallisation trials. Buffer screens were also performed on the USP20 1-101 construct to increase its stability for crystallisation. Monodisperse, pure protein of any catalytic domain-containing construct of USP20 was unobtainable; only a trigger factor-tagged full length USP20 was purified and active. Two constructs containing the double DUSP domains were produced (USP20 686-914 and 686-894), and both suffered from a low solubility limit. Buffer screening was used to increase its stability, which identified ethylene glycol as a stabilising additive. Due to the nature of commonly used solubility tags, novel tags were designed that would potentially benefit the crystallisation of the fusion construct. Identified from the PDB and literature searches, the calponin homology domain from human β-spectrin (PDB code 1BKR) and the receiver domain from Myxococcus xanthus social motility protein frzS (PDB code 2GKG) were used. Both new tags, as well as MBP were fused to the N-terminus of the DUSP domains (USP20 686-894) to enhance solubility and crystallise the DUSP domains. 2GKG was an effective solubility tag, increased the solubility of the DUSP domains to near that of the MBP fusion. 1BKR, however, was only marginally useful as a solubility tag. In total 97 crystallisation trials were set up for all constructs of USP20, but no crystals containing USP20 protein formed. Y2H assays were used to investigate the interaction between USP20 domains and Β-arrestin-1, TRAF6, RAD17 and PLK1. Of these, only and interaction between USP20’s DUSP domains (residues 686-894) and full length PLK1 was observed. Interestingly, further Y2H and ELISA showed a non-canonical, binary interaction between the poloboxes of PLK1 (residues 367-603) and the DUSP domains. Pull down assays produced a list of possible novel interactors for USP20. These include proteins implicated in processes known, and unknown, to involve USP20. Finally, using ELISA, thermal shift assays and ITC, it was shown that the zinc finger domain of USP20 does not bind to ubiquitin

An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system

The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.</p

The structure of the deubiquitinase USP15 reveals a misaligned catalytic triad and an open ubiquitin-binding channel

© 2018 Ward et al. Ubiquitin-specific protease 15 (USP15) regulates important cellular processes, including transforming growth factor β (TGF-β) signaling, mitophagy, mRNA processing, and innate immune responses; however, structural information on USP15's catalytic domain is currently unavailable. Here, we determined crystal structures of the USP15 catalytic core domain, revealing a canonical USP fold, including a finger, palm, and thumb region. Unlike for the structure of paralog USP4, the catalytic triad is in an inactive configuration with the catalytic cysteine ∼10 Å apart from the catalytic histidine. This conformation is atypical, and a similar misaligned catalytic triad has so far been observed only for USP7, although USP15 and USP7 are differently regulated. Moreover, we found that the active-site loops are flexible, resulting in a largely open ubiquitin tail-binding channel. Comparison of the USP15 and USP4 structures points to a possible activation mechanism. Sequence differences between these two USPs mainly map to the S1' region likely to confer specificity, whereas the S1 ubiquitin-binding pocket is highly conserved. Isothermal titration calorimetry monoubiquitin- and linear diubiquitin-binding experiments showed significant differences in their thermodynamic profiles, with USP15 displaying a lower affinity for monoubiquitin than USP4. Moreover, we report that USP15 is weakly inhibited by the antineoplastic agent mitoxantrone in vitro A USP15-mitoxantrone complex structure disclosed that the anthracenedione interacts with the S1' binding site. Our results reveal first insights into USP15's catalytic domain structure, conformational changes, differences between paralogs, and small-molecule interactions and establish a framework for cellular probe and inhibitor development

Discovery of peptide ligands targeting a specific ubiquitin-like domain– binding site in the deubiquitinase USP11

© 2019 Spiliotopoulos et al. Published by The American Society for Biochemistry and Molecular Biology, Inc. Ubiquitin-specific proteases (USPs) reverse ubiquitination and regulate virtually all cellular processes. Defined noncatalytic domains in USP4 and USP15 are known to interact with E3 ligases and substrate recruitment factors. No such interactions have been reported for these domains in the paralog USP11, a key regulator of DNA double-strand break repair by homologous recombination. We hypothesized that USP11 domains adjacent to its protease domain harbor unique peptide-binding sites. Here, using a next-generation phage display (NGPD) strategy, combining phage display library screening with next-generation sequencing, we discovered unique USP11-interacting peptide motifs. Isothermal titration calorimetry disclosed that the highest affinity peptides (K D of 10 M) exhibit exclusive selectivity for USP11 over USP4 and USP15 in vitro. Furthermore, a crystal structure of a USP11–peptide complex revealed a previously unknown binding site in USP11’s noncatalytic ubiquitin-like (UBL) region. This site interacted with a helical motif and is absent in USP4 and USP15. Reporter assays using USP11-WT versus a binding pocket– deficient double mutant disclosed that this binding site modulates USP11’s function in homologous recombination–mediated DNA repair. The highest affinity USP11 peptide binder fused to a cellular delivery sequence induced significant nuclear localization and cell cycle arrest in S phase, affecting the viability of different mammalian cell lines. The USP11 peptide ligands and the paralog-specific functional site in USP11 identified here provide a framework for the development of new biochemical tools and therapeutic agents. We propose that an NGPD-based strategy for identifying interacting peptides may be applied also to other cellular targets

Structural basis of the leukocyte integrin Mac-1 I-domain interactions with the platelet glycoprotein Ib

Cell-surface receptor interactions between leukocyte integrin macrophage-1 antigen (Mac-1, also known as CR3, aMb2, CD11b/CD18) and platelet glycoprotein Iba (GPIba) are critical to vascular in?ammation. To de?ne the key residues at the binding interface, we used nuclear magnetic resonance (NMR) to assign the spectra of the mouse Mac-1 I-domain and mapped the residues contacting the mouse GPIba N-terminal domain (GPIbaN) to the locality of the integrin metal ion-dependant adhesion site (MIDAS) surface. We next determined the crystal structures of the mouse GPIbaN and Mac-1 I-domain to 2 ?A and 2.5 ?A resolution, respectively. The mouse Mac-1 I-domain crystal structure reveals an active conformation that is stabilized by a crystal contact from the a7-helix with a glutamatesidechaincompletingtheoctahedralcoordinationsphereoftheMIDASMg21 ion. The amino acid sequence of the a7-helix and disposition of the glutamic acid matches the C-terminal capping region a-helix of GPIba effectively acting as a ligand mimetic. Using these crystal structures in combination with NMR measurements and docking analysis, we developed a model whereby an acidic residue from the GPIba leucine-rich repeat (LRR) capping a-helix coordinates directly to the Mac-1 MIDAS Mg21 ion. The Mac-1:GPIbaN complex involves additional interactions consolidated by an elongated pocket ?anking the GPIbaN LRR capping a-helix. The GPIbaN a-helix has an HxxxE motif, which is equivalent by homology to RxxxD from the human GPIbaN. Subsequent mutagenesis of residues at this interface, coupled with surface plasmon resonance studies, con?rmed the importance of GPIbaN residues H218, E222, and the Mac-1 MIDAS residue T209 to formation of the complex

Protein target highlights in CASP15: Analysis of models by structure providers

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology

Characterisation of the deubiquitinating enzyme USP20

USP20 is a deubiquitinating enzyme that is involved in a number of important cellular pathways, including thyroid metabolism, hypoxic response, seven transmembrane receptor signalling, NF-κβ signalling, centrosome homeostasis and DNA repair. Of recent, it is becoming a major deubiquitinase involved in regulating the DNA-damage response pathway and cell cycle checkpoints. The protein consists of a zinc finger domain, catalytic domain and two ‘domain present in USP’ (DUSP) domains; an architecture shared only with its paralogue USP33. There is no structural information on any of the domains of USP20, so crystallisation trials of the domains of USP20 were performed in order to solve their structures by X-ray crystallography. In addition, yeast two-hybrid (Y2H) and in vitro assays were used to further characterise known and putative interactors of USP20. Finally, the zinc finger domain and DUSP domains were used in pull down assays to identify USP20-interacting proteins from HEK293 lysate. Two stable and well-expressing constructs of the zing finger domain (USP20 1-101 and 1-108) were purified and set up for crystallisation trials. Buffer screens were also performed on the USP20 1-101 construct to increase its stability for crystallisation. Monodisperse, pure protein of any catalytic domain-containing construct of USP20 was unobtainable; only a trigger factor-tagged full length USP20 was purified and active. Two constructs containing the double DUSP domains were produced (USP20 686-914 and 686-894), and both suffered from a low solubility limit. Buffer screening was used to increase its stability, which identified ethylene glycol as a stabilising additive. Due to the nature of commonly used solubility tags, novel tags were designed that would potentially benefit the crystallisation of the fusion construct. Identified from the PDB and literature searches, the calponin homology domain from human β-spectrin (PDB code 1BKR) and the receiver domain from Myxococcus xanthus social motility protein frzS (PDB code 2GKG) were used. Both new tags, as well as MBP were fused to the N-terminus of the DUSP domains (USP20 686-894) to enhance solubility and crystallise the DUSP domains. 2GKG was an effective solubility tag, increased the solubility of the DUSP domains to near that of the MBP fusion. 1BKR, however, was only marginally useful as a solubility tag. In total 97 crystallisation trials were set up for all constructs of USP20, but no crystals containing USP20 protein formed. Y2H assays were used to investigate the interaction between USP20 domains and Β-arrestin-1, TRAF6, RAD17 and PLK1. Of these, only and interaction between USP20’s DUSP domains (residues 686-894) and full length PLK1 was observed. Interestingly, further Y2H and ELISA showed a non-canonical, binary interaction between the poloboxes of PLK1 (residues 367-603) and the DUSP domains. Pull down assays produced a list of possible novel interactors for USP20. These include proteins implicated in processes known, and unknown, to involve USP20. Finally, using ELISA, thermal shift assays and ITC, it was shown that the zinc finger domain of USP20 does not bind to ubiquitin