I03-Macromolecular Crystallography
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Paul R.
Elliott
,
Derek
Leske
,
Jane
Wagstaff
,
Lisa
Schlicher
,
Georgina
Berridge
,
Sarah
Maslen
,
Frederik
Timmermann
,
Biao
Ma
,
Roman
Fischer
,
Stefan M. V.
Freund
,
David
Komander
,
Mads
Gyrd-Hansen
Open Access
Abstract: Non-degradative ubiquitin chains and phosphorylation events govern signaling responses by innate immune receptors. The deubiquitinase CYLD in complex with SPATA2 is recruited to receptor signaling complexes by the ubiquitin ligase LUBAC and regulates Met1- and Lys63-linked polyubiquitin and receptor signaling outcomes. Here, we investigate the molecular determinants of CYLD activity. We reveal that two CAP-Gly domains in CYLD are ubiquitin-binding domains and demonstrate a requirement of CAP-Gly3 for CYLD activity and regulation of immune receptor signaling. Moreover, we identify a phosphorylation switch outside of the catalytic USP domain, which activates CYLD toward Lys63-linked polyubiquitin. The phosphorylated residue Ser568 is a novel tumor necrosis factor (TNF)-regulated phosphorylation site in CYLD and works in concert with Ser418 to enable CYLD-mediated deubiquitination and immune receptor signaling. We propose that phosphorylated CYLD, together with SPATA2 and LUBAC, functions as a ubiquitin-editing complex that balances Lys63- and Met1-linked polyubiquitin at receptor signaling complexes to promote LUBAC signaling.
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Oct 2021
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I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Alexander F.
Schubert
,
Justine V
Nguyen
,
Tyler G.
Franklin
,
Paul P.
Geurink
,
Cameron G.
Roberts
,
Daniel J.
Sanderson
,
Lauren N.
Miller
,
Huib
Ovaa
,
Kay
Hofmann
,
Jonathan N.
Pruneda
,
David
Komander
Diamond Proposal Number(s):
[8547, 11235]
Open Access
Abstract: Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, invading pathogens can inactivate immune response pathways and evade detection. The ovarian tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans. Viral pathogens have also extensively co‐opted the OTU fold to subvert host signaling, but the extent to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. By determining the ubiquitin‐bound structures of two examples, we demonstrate the novel strategies that have evolved to both thread an OTU fold and recognize a ubiquitin substrate. With these new examples, we perform the first cross‐kingdom structural analysis of the OTU fold that highlights commonalities among distantly related OTU deubiquitinases.
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Jun 2020
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I03-Macromolecular Crystallography
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Aurelia
Stangl
,
Paul R.
Elliott
,
Adan
Pinto-Fernandez
,
Sarah
Bonham
,
Luke
Harrison
,
Annalisa
Schaub
,
Kerstin
Kutzner
,
Kirstin
Keusekotten
,
Paul T.
Pfluger
,
Farid
El Oualid
,
Benedikt M.
Kessler
,
David
Komander
,
Daniel
Krappmann
Open Access
Abstract: OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association, OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the VPS26A-retromer subunit and endosome-to-plasma membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of SNX27-retromer assembly and recycling to the cell surface.
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Sep 2019
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B21-High Throughput SAXS
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Open Access
Abstract: The evolutionarily related deubiquitinating enzymes (DUBs) USP25 and USP28 comprise an identical overall domain architecture but are functionally non-redundant: USP28 stabilizes c-MYC and other nuclear proteins, and USP25 regulates inflammatory TRAF signaling. We here compare molecular features of USP25 and USP28. Active enzymes form distinctively shaped dimers, with a dimerizing insertion spatially separating independently active catalytic domains. In USP25, but not USP28, two dimers can form an autoinhibited tetramer, where a USP25-specific, conserved insertion sequence blocks ubiquitin binding. In full-length enzymes, a C-terminal domain with a previously unknown fold has no impact on oligomerization, but N-terminal regions affect the dimer-tetramer equilibrium in vitro. We confirm oligomeric states of USP25 and USP28 in cells and show that modulating oligomerization affects substrate stabilization in accordance with in vitro activity data. Our work highlights how regions outside of the catalytic domain enable a conceptually intriguing interplay of DUB oligomerization and activity.
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Mar 2019
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I03-Macromolecular Crystallography
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Open Access
Abstract: The deubiquitinase OTULIN removes methionine‐1 (M1)‐linked polyubiquitin signals conjugated by the linear ubiquitin chain assembly complex (LUBAC) and is critical for preventing TNF‐driven inflammation in OTULIN‐related autoinflammatory syndrome (ORAS). Five ORAS patients have been reported, but how dysregulated M1‐linked polyubiquitin signalling causes their symptoms is unclear. Here, we report a new case of ORAS in which an OTULIN‐Gly281Arg mutation leads to reduced activity and stability in vitro and in cells. In contrast to OTULIN‐deficient monocytes, in which TNF signalling and NF‐κB activation are increased, loss of OTULIN in patient‐derived fibroblasts leads to a reduction in LUBAC levels and an impaired response to TNF. Interestingly, both patient‐derived fibroblasts and OTULIN‐deficient monocytes are sensitised to certain types of TNF‐induced death, and apoptotic cells are evident in ORAS patient skin lesions. Remarkably, haematopoietic stem cell transplantation leads to complete resolution of inflammatory symptoms, including fevers, panniculitis and diarrhoea. Therefore, haematopoietic cells are necessary for clinical manifestation of ORAS. Together, our data suggest that ORAS pathogenesis involves hyper‐inflammatory immune cells and TNF‐induced death of both leukocytes and non‐haematopoietic cells.
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Feb 2019
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I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8547, 11235]
Abstract: Pathogenic bacteria are armed with potent effector proteins that subvert host signalling processes during infection1. The activities of bacterial effectors and their associated roles within the host cell are often poorly understood, particularly for Chlamydia trachomatis2, a World Health Organization designated neglected disease pathogen. We identify and explain remarkable dual Lys63-deubiquitinase (DUB) and Lys-acetyltransferase activities in the Chlamydia effector ChlaDUB1. Crystal structures capturing intermediate stages of each reaction reveal how the same catalytic centre of ChlaDUB1 can facilitate such distinct processes, and enable the generation of mutations that uncouple the two activities. Targeted Chlamydia mutant strains allow us to link the DUB activity of ChlaDUB1 and the related, dedicated DUB ChlaDUB2 to fragmentation of the host Golgi apparatus, a key process in Chlamydia infection for which effectors have remained elusive. Our work illustrates the incredible versatility of bacterial effector proteins, and provides important insights towards understanding Chlamydia pathogenesis.
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Nov 2018
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[15916]
Abstract: Mutations in the E3 ubiquitin ligase parkin (PARK2) and the protein kinase PINK1 (PARK6) are linked to autosomal-recessive juvenile Parkinsonism (AR-JP)1,2, and at the cellular level cause defects in mitophagy, the cellular process that organises destruction of damaged mitochondria3,4. Parkin is autoinhibited, and requires activation by PINK1, which phosphorylates Ser65 in ubiquitin and in the parkin ubiquitin-like (Ubl) domain. Parkin binds phospho-ubiquitin, which enables efficient Parkin phosphorylation; however, the enzyme remains autoinhibited with an inaccessible active site5,6. It is unclear how phosphorylation of parkin activates the molecule. Here we follow the activation of full-length human parkin by hydrogen deuterium exchange mass spectrometry, and reveal large-scale domain rearrangement in the activation process, in which the phospho-Ubl rebinds to the parkin core, and releases the catalytic RING2 domain. A 1.8 Å crystal structure of phosphorylated human parkin reveals the binding site of the phosphorylated Ubl on the unique parkin domain (UPD), involving a phosphate-binding pocket lined by AR-JP mutations. Strikingly, a conserved linker region between Ubl and UPD acts as an activating element (ACT) that contributes to RING2 release by mimicking RING2 interactions on the UPD, explaining further AR-JP mutations. Our data unveil how autoinhibition in parkin is resolved, and suggest how parkin ubiquitinates its substrates via an untethered RING2 domain. This opens exciting new avenues to design parkin activators for clinical use.
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Jun 2018
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Kirby N.
Swatek
,
Martina
Aumayr
,
Jonathan N.
Pruneda
,
Linda J.
Visser
,
Stephen
Berryman
,
Anja F.
Kueck
,
Paul P.
Geurink
,
Huib
Ovaa
,
Frank J. M.
Van Kuppeveld
,
Tobias J.
Tuthill
,
Tim
Skern
,
David
Komander
Diamond Proposal Number(s):
[11235]
Open Access
Abstract: In response to viral infection, cells mount a potent inflammatory response that relies on ISG15 and ubiquitin posttranslational modifications. Many viruses use deubiquitinases and deISGylases that reverse these modifications and antagonize host signaling processes. We here reveal that the leader protease, Lbpro, from foot-and-mouth disease virus (FMDV) targets ISG15 and to a lesser extent, ubiquitin in an unprecedented manner. Unlike canonical deISGylases that hydrolyze the isopeptide linkage after the C-terminal GlyGly motif, Lbpro cleaves the peptide bond preceding the GlyGly motif. Consequently, the GlyGly dipeptide remains attached to the substrate Lys, and cleaved ISG15 is rendered incompetent for reconjugation. A crystal structure of Lbpro bound to an engineered ISG15 suicide probe revealed the molecular basis for ISG15 proteolysis. Importantly, anti-GlyGly antibodies, developed for ubiquitin proteomics, are able to detect Lbpro cleavage products during viral infection. This opens avenues for infection detection of FMDV based on an immutable, host-derived epitope.
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Feb 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[15916]
Abstract: The Ser/Thr protein kinase PINK1 phosphorylates the well‐folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We previously showed that Ser65 phosphorylation results in a conformational change in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation wherein the last β‐strand is retracted to extend the Ser65 loop and shorten the C‐terminal tail. We show using chemical exchange saturation transfer (CEST) nuclear magnetic resonance experiments that a similar, C‐terminally retracted (Ub‐CR) conformation also exists at low population in wild‐type Ub. Point mutations in the moving β5 and neighbouring β‐strands shift the Ub/Ub‐CR equilibrium. This enabled functional studies of the two states, and we show that while the Ub‐CR conformation is defective for conjugation, it demonstrates improved binding to PINK1 through its extended Ser65 loop, and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub‐CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded protein domains.
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Nov 2017
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
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Abstract: Autosomal recessive juvenile Parkinsonism (AR-JP) is caused by mutations in a number of PARK genes, in particular in the E3 ubiquitin ligase Parkin (PARK2), and in its upstream protein kinase PINK1 (PARK6). PINK1 phosphorylates ubiquitin and the Parkin ubiquitin-like domain on structurally protected Ser65 to trigger mitophagy. We here report a crystal structure of a nanobody-stabilised complex between Pediculus humanus corporis (Ph)PINK1 with ubiquitin in the ‘C-terminally retracted’ (Ub-CR) conformation. The structure reveals many peculiarities of PINK1, including the architecture of the C-terminal region, and reveals how the PINK1 N-lobe binds ubiquitin via a unique insertion. The flexible Ser65-loop in the Ub-CR conformation reaches the activation segment, facilitating placement of Ser65 in a phosphate accepting position. The structure further explains how autophosphorylation in the N-lobe stabilises structurally and functionally important insertions, and reveals the molecular basis for AR-JP causing mutations, some of which disrupt ubiquitin binding.
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Oct 2017
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