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17 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	A tri-ionic anchor mechanism drives Ube2N-specific recruitment and K63-chain ubiquitination in TRIM ligases Leo Kiss , Jingwei Zeng , Claire Dickson , Donna L. Mallery , Ji-chun Yang , Stephen H. Mclaughlin , Andreas Boland , David Neuhaus , Leo C. James Nature Communications 10 DOI: 10.1038/s41467-019-12388-y Diamond Proposal Number(s): [15916] Open Access Show Abstract ▼ Abstract: The cytosolic antibody receptor TRIM21 possesses unique ubiquitination activity that drives broad-spectrum anti-pathogen targeting and underpins the protein depletion technology Trim-Away. This activity is dependent on formation of self-anchored, K63-linked ubiquitin chains by the heterodimeric E2 enzyme Ube2N/Ube2V2. Here we reveal how TRIM21 facilitates ubiquitin transfer and differentiates this E2 from other closely related enzymes. A tri-ionic motif provides optimally distributed anchor points that allow TRIM21 to wrap an Ube2N~Ub complex around its RING domain, locking the closed conformation and promoting ubiquitin discharge. Mutation of these anchor points inhibits ubiquitination with Ube2N/Ube2V2, viral neutralization and immune signalling. We show that the same mechanism is employed by the anti-HIV restriction factor TRIM5 and identify spatially conserved ionic anchor points in other Ube2N-recruiting RING E3s. The tri-ionic motif is exclusively required for Ube2N but not Ube2D1 activity and provides a generic E2-specific catalysis mechanism for RING E3s.	Oct 2019
I03-Macromolecular Crystallography Krios I-Titan Krios I at Diamond	The structure of bactofilin filaments reveals their mode of membrane binding and lack of polarity Xian Deng , Andres Gonzalez Llamazares , James Wagstaff , Victoria L. Hale , Giuseppe Cannone , Stephen H. Mclaughlin , Danguole Kureisaite-ciziene , Jan Lowe Nature Microbiology 16 DOI: 10.1038/s41564-019-0544-0 Show Abstract ▼ Abstract: Bactofilins are small β-helical proteins that form cytoskeletal filaments in a range of bacteria. Bactofilins have diverse functions, from cell stalk formation in Caulobacter crescentus to chromosome segregation and motility in Myxococcus xanthus. However, the precise molecular architecture of bactofilin filaments has remained unclear. Here, sequence analysis and electron microscopy results reveal that, in addition to being widely distributed across bacteria and archaea, bactofilins are also present in a few eukaryotic lineages such as the Oomycetes. Electron cryomicroscopy analysis demonstrated that the sole bactofilin from Thermus thermophilus (TtBac) forms constitutive filaments that polymerize through end-to-end association of the β-helical domains. Using a nanobody, we determined the near-atomic filament structure, showing that the filaments are non-polar. A polymerization-impairing mutation enabled crystallization and structure determination, while reaffirming the lack of polarity and the strength of the β-stacking interface. To confirm the generality of the lack of polarity, we performed coevolutionary analysis on a large set of sequences. Finally, we determined that the widely conserved N-terminal disordered tail of TtBac is responsible for direct binding to lipid membranes, both on liposomes and in Escherichia coli cells. Membrane binding is probably a common feature of these widespread but only recently discovered filaments of the prokaryotic cytoskeleton.	Sep 2019
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	An oligomeric state‐dependent switch in the ER enzyme FICD regulates AMP ylation and de AMP ylation of BiP Luke A. Perera , Claudia Rato , Yahui Yan , Lisa Neidhardt , Stephen H. Mclaughlin , Randy J. Read , Steffen Preissler , David Ron The Embo Journal 66 DOI: 10.15252/embj.2019102177 Diamond Proposal Number(s): [15916] Open Access Show Abstract ▼ Abstract: AMPylation is an inactivating modification that alters the activity of the major endoplasmic reticulum (ER) chaperone BiP to match the burden of unfolded proteins. A single ER‐localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD's activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface, or of residues along an inhibitory pathway linking the dimer interface to the enzyme's active site, favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation‐competent binding of MgATP. Moreover, a reciprocal signal, propagated from the nucleotide‐binding site, provides a mechanism for coupling the oligomeric state and enzymatic activity of FICD to the energy status of the ER.	Sep 2019
I03-Macromolecular Crystallography	Structure of KAP1 tripartite motif identifies molecular interfaces required for retroelement silencing Guido A. Stoll , Shun-ichiro Oda , Zheng-shan Chong , Minmin Yu , Stephen H. Mclaughlin , Yorgo Modis Proceedings Of The National Academy Of Sciences 60 DOI: 10.1073/pnas.1901318116 Diamond Proposal Number(s): [15916] Open Access Show Abstract ▼ Abstract: Retroviruses can integrate their DNA into the host-cell genome. Inherited retroviral DNA and other transposable elements account for more than half of the human genome. Transposable elements must be tightly regulated to restrict their proliferation and prevent toxic gene expression. KAP1/TRIM28 is an essential regulator of transposable element transcription. We determined the crystal structure of the KAP1 TRIM. The structure identifies a protein–protein interaction site required for recruitment of KAP1 to transposable elements. An epigenetic gene silencing assay confirms the importance of this site for KAP1-dependent silencing. We also show that KAP1 self-assembles in solution, but this self-assembly is not required for silencing. Our work provides insights into KAP1-dependent silencing and tools for expanding our mechanistic understanding of this process.	Jul 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Structural analysis of the interaction between the bacterial cell division proteins FtsQ and FtsB Danguole Kureisaite-ciziene , Aravindan Varadajan , Stephen H. Mclaughlin , Marjolein Glas , Alejandro Montón Silva , Rosa Luirink , Carolin Mueller , Tanneke Den Blaauwen , Tom N. Grossmann , Joen Luirink , Jan Lowe Mbio 9 DOI: 10.1128/mBio.01346-18 Open Access Show Abstract ▼ Abstract: Most bacteria and archaea use the tubulin homologue FtsZ as its central organizer of cell division. In Gram-negative Escherichia coli bacteria, FtsZ recruits cytosolic, transmembrane, periplasmic, and outer membrane proteins, assembling the divisome that facilitates bacterial cell division. One such divisome component, FtsQ, a bitopic membrane protein with a globular domain in the periplasm, has been shown to interact with many other divisome proteins. Despite its otherwise unknown function, it has been shown to be a major divisome interaction hub. Here, we investigated the interactions of FtsQ with FtsB and FtsL, two small bitopic membrane proteins that act immediately downstream of FtsQ. We show in biochemical assays that the periplasmic domains of E. coli FtsB and FtsL interact with FtsQ, but not with each other. Our crystal structure of FtsB bound to the β domain of FtsQ shows that only residues 64 to 87 of FtsB interact with FtsQ. A synthetic peptide comprising those 24 FtsB residues recapitulates the FtsQ-FtsB interactions. Protein deletions and structure-guided mutant analyses validate the structure. Furthermore, the same structure-guided mutants show cell division defects in vivo that are consistent with our structure of the FtsQ-FtsB complex that shows their interactions as they occur during cell division. Our work provides intricate details of the interactions within the divisome and also provides a tantalizing view of a highly conserved protein interaction in the periplasm of bacteria that is an excellent target for cell division inhibitor searches.	Sep 2018
I02-Macromolecular Crystallography I03-Macromolecular Crystallography	A low-complexity region in the YTH domain protein Mmi1 enhances RNA binding James A. W. Stowell , Jane L. Wagstaff , Chris H. Hill , Minmin Yu , Stephen H. Mclaughlin , Stefan M. V. Freund , Lori A. Passmore Journal Of Biological Chemistry DOI: 10.1074/jbc.RA118.002291 Diamond Proposal Number(s): [8547, 11235] Show Abstract ▼ Abstract: Mmi1 is an essential RNA-binding protein in the fission yeast Schizosaccharomyces pombe that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold. Here, we report that an N-terminal extension that is proximal to the YTH domain enhances RNA binding. Using X-ray crystallography, NMR and biophysical methods, we show that this low-complexity region becomes more ordered upon RNA binding. This enhances the affinity of the interaction of the Mmi1 YTH domain with specific RNAs by reducing the dissociation rate of the Mmi1–RNA complex. We propose that the low-complexity region influences RNA binding indirectly by reducing dynamic motions of the RNA binding groove and stabilizing a conformation of the YTH domain that binds to RNA with high affinity. Taken together, our work reveals how a low-complexity region proximal to a conserved folded domain can adopt an ordered structure to aid nucleic acid binding.	Apr 2018
I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis for the dimerization of Nab2 generated by RNA binding provides insight into its contribution to both poly(A) tail length determination and transcript compaction in Saccharomyces cerevisiae Shintaro Aibara , James M. B. Gordon , Anja S. Riesterer , Stephen H. Mclaughlin , Murray Stewart Nucleic Acids Research DOI: 10.1093/nar/gkw1224 Open Access Show Abstract ▼ Abstract: In Saccharomyces cerevisiae generation of export-competent mRNPs terminates the nuclear phase of the gene expression pathway and facilitates transport to the cytoplasm for translation. Nab2 functions in this process to control both mRNP compaction that facilitates movement through nuclear pore complexes and the length of transcript poly(A) tails. Nab2 has a modular structure that includes seven CCCH Zn fingers that bind to A-rich RNAs and fingers 5–7 are critical for these functions. Here, we demonstrate, using both biophysical and structural methods, that binding A11G RNA induces dimerization of Zn fingers 5–7 mediated by the novel spatial arrangement of the fingers promoting each RNA chain binding two protein chains. The dimerization of Nab2 induced by RNA binding provides a basis for understanding its function in both poly(A) tail length regulation and in the compaction of mature transcripts to facilitate nuclear export.	Dec 2016
I04-Macromolecular Crystallography	The Ciliopathy-Associated Cep104 Protein Interacts with Tubulin and Nek1 Kinase Caezar Al-jassar , Antonina Andreeva , Deepak D. Barnabas , Stephen H. Mclaughlin , Christopher M. Johnson , Minmin Yu , Mark Van Breugel Structure DOI: 10.1016/j.str.2016.11.014 Diamond Proposal Number(s): [11235] Open Access Show Abstract ▼ Abstract: Cilia are thin cell projections with essential roles in cell motility, fluid movement, sensing, and signaling. They are templated from centrioles that dock against the plasma membrane and subsequently extend their peripheral microtubule array. The molecular mechanisms underpinning cilia assembly are incompletely understood. Cep104 is a key factor involved in cilia formation and length regulation that rides on the ends of elongating and shrinking cilia. It is mutated in Joubert syndrome, a genetically heterogeneous ciliopathy. Here we provide structural and biochemical data that Cep104 contains a tubulin-binding TOG (tumor overexpressed gene) domain and a novel C2HC zinc finger array. Furthermore, we identify the kinase Nek1, another ciliopathy-associated protein, as a potential binding partner of this array. Finally, we show that Nek1 competes for binding to Cep104 with the distal centriole-capping protein CP110. Our data suggest a model for Cep104 activity during ciliogenesis and provide a novel link between Cep104 and Nek1.	Dec 2016
I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Crenactin forms actin-like double helical filaments regulated by arcadin-2 Thierry Izore , Danguole Kureisaite-ciziene , Stephen H Mclaughlin , Jan Lowe Elife 5 DOI: 10.7554/eLife.21600 Diamond Proposal Number(s): [11235] Open Access Show Abstract ▼ Abstract: The similarity of eukaryotic actin to crenactin, a filament-forming protein from the crenarchaeon Pyrobaculum calidifontis supports the theory of a common origin of Crenarchaea and Eukaryotes. Monomeric structures of crenactin and actin are similar, although their filament architectures were suggested to be different. Here we report that crenactin forms bona fide double helical filaments that show exceptional similarity to eukaryotic F-actin. With cryo-electron microscopy and helical reconstruction we solved the structure of the crenactin filament to 3.8 Å resolution. When forming double filaments, the 'hydrophobic plug' loop in crenactin rearranges. Arcadin-2, also encoded by the arcade gene cluster, binds tightly with its C-terminus to the hydrophobic groove of crenactin. Binding is reminiscent of eukaryotic actin modulators such as cofilin and thymosin β4 and arcadin-2 is a depolymeriser of crenactin filaments. Our work further supports the theory of shared ancestry of Eukaryotes and Crenarchaea.	Nov 2016
I02-Macromolecular Crystallography	SPATA2 Links CYLD to LUBAC, Activates CYLD, and Controls LUBAC Signaling Paul R. Elliott , Derek Leske , Matous Hrdinka , Katrin Bagola , Berthe k. Fiil , Stephen h. Mclaughlin , Jane Wagstaff , Norbert Volkmar , John c. Christianson , Benedikt m. Kessler , Stefan m. V. Freund , David Komander , Mads Gyrd-hansen Molecular Cell 63, 990 - 1005 DOI: 10.1016/j.molcel.2016.08.001 Diamond Proposal Number(s): [11235] Open Access Show Abstract ▼ Abstract: The linear ubiquitin chain assembly complex (LUBAC) regulates immune signaling, and its function is regulated by the deubiquitinases OTULIN and CYLD, which associate with the catalytic subunit HOIP. However, the mechanism through which CYLD interacts with HOIP is unclear. We here show that CYLD interacts with HOIP via spermatogenesis-associated protein 2 (SPATA2). SPATA2 interacts with CYLD through its non-canonical PUB domain, which binds the catalytic CYLD USP domain in a CYLD B-box-dependent manner. Significantly, SPATA2 binding activates CYLD-mediated hydrolysis of ubiquitin chains. SPATA2 also harbors a conserved PUB-interacting motif that selectively docks into the HOIP PUB domain. In cells, SPATA2 is recruited to the TNF receptor 1 signaling complex and is required for CYLD recruitment. Loss of SPATA2 increases ubiquitination of LUBAC substrates and results in enhanced NOD2 signaling. Our data reveal SPATA2 as a high-affinity binding partner of CYLD and HOIP, and a regulatory component of LUBAC-mediated NF-κB signaling.	Sep 2016

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