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25 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
Krios IV-Titan Krios IV at Diamond	Fibrillin microfibril structure identifies long-range effects of inherited pathogenic mutations affecting a key regulatory latent TGFβ-binding site Alan R. F. Godwin , Rana Dajani , Xinyang Zhang , Jennifer Thomson , David F. Holmes , Christin S. Adamo , Gerhard Sengle , Michael J. Sherratt , Alan M. Roseman , Clair Baldock Nature Structural & Molecular Biology 84 DOI: 10.1038/s41594-023-00950-8 Diamond Proposal Number(s): [16619] Open Access Show Abstract ▼ Abstract: Genetic mutations in fibrillin microfibrils cause serious inherited diseases, such as Marfan syndrome and Weill–Marchesani syndrome (WMS). These diseases typically show major dysregulation of tissue development and growth, particularly in skeletal long bones, but links between the mutations and the diseases are unknown. Here we describe a detailed structural analysis of native fibrillin microfibrils from mammalian tissue by cryogenic electron microscopy. The major bead region showed pseudo eightfold symmetry where the amino and carboxy termini reside. On the basis of this structure, we show that a WMS deletion mutation leads to the induction of a structural rearrangement that blocks interaction with latent TGFβ-binding protein-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. The integrin αvβ3-binding site was also mapped onto the microfibril structure. These results establish that in complex extracellular assemblies, such as fibrillin microfibrils, mutations may have long-range structural consequences leading to the disruption of growth factor signaling and the development of disease.	Apr 2023
B21-High Throughput SAXS Krios IV-Titan Krios IV at Diamond	Structure of PLA2R reveals presentation of the dominant membranous nephropathy epitope and an immunogenic patch Maryline Fresquet , Michael P. Lockhart-Cairns , Samuel J. Rhoden , Thomas A. Jowitt , David C. Briggs , Clair Baldock , Paul E. Brenchley , Rachel Lennon Proceedings Of The National Academy Of Sciences 119 DOI: 10.1073/pnas.2202209119 Diamond Proposal Number(s): [17773, 22724] Open Access Show Abstract ▼ Abstract: Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most prevalent circulating autoantibodies in membranous nephropathy are against phospholipase A2 receptor (PLA2R), a cell surface receptor. The dominant epitope in PLA2R is located within the cysteine-rich domain, yet high-resolution structure-based mapping is lacking. In this study, we define the key nonredundant amino acids in the dominant epitope of PLA2R involved in autoantibody binding. We further describe two essential regions within the dominant epitope and spacer requirements for a synthetic peptide of the epitope for drug discovery. In addition, using cryo-electron microscopy, we have determined the high-resolution structure of PLA2R to 3.4 Å resolution, which shows that the dominant epitope and key residues within the cysteine-rich domain are accessible at the cell surface. In addition, the structure of PLA2R not only suggests a different orientation of domains but also implicates a unique immunogenic signature in PLA2R responsible for inducing autoantibody formation and recognition.	Jul 2022
B21-High Throughput SAXS	Autosomal recessive cutis Laxa 1C mutations disrupt the structure and interactions of latent TGFβ binding protein-4 Yasmene F. Alanazi , Michael P. Lockhart-Cairns , Stuart A. Cain , Thomas A. Jowitt , Anthony S. Weiss , Clair Baldock Frontiers In Genetics 12 DOI: 10.3389/fgene.2021.706662 Diamond Proposal Number(s): [1580] Open Access Show Abstract ▼ Abstract: Latent TGFβ binding protein-4 (LTBP4) is a multi-domain glycoprotein, essential for regulating the extracellular bioavailability of TGFβ and assembly of elastic fibre proteins, fibrillin-1 and tropoelastin. LTBP4 mutations are linked to autosomal recessive cutis laxa type 1C (ARCL1C), a rare congenital disease characterised by high mortality and severely disrupted connective tissues. Despite the importance of LTBP4, the structure and molecular consequences of disease mutations are unknown. Therefore, we analysed the structural and functional consequences of three ARCL1C causing point mutations which effect highly conserved cysteine residues. Our structural and biophysical data show that the LTBP4 N- and C-terminal regions are monomeric in solution and adopt extended conformations with the mutations resulting in subtle changes to their conformation. Similar to LTBP1, the N-terminal region is relatively inflexible, whereas the C-terminal region is flexible. Interaction studies show that one C-terminal mutation slightly decreases binding to fibrillin-1. We also found that the LTBP4 C-terminal region directly interacts with tropoelastin which is perturbed by both C-terminal ARCL1C mutations, whereas an N-terminal mutation increased binding to fibulin-4 but did not affect the interaction with heparan sulphate. Our results suggest that LTBP4 mutations contribute to ARCL1C by disrupting the structure and interactions of LTBP4 which are essential for elastogenesis in a range of mammalian connective tissues.	Sep 2021
B21-High Throughput SAXS	Priming human elastic proteins for assembly into elastic tissues Clair Baldock Diamond Proposal Number(s): [17773] Show Abstract ▼ Abstract: Elastic fibres are the main elastic component of mammalian elastic tissues such as major arteries, the lungs and skin. They provide resilience and recoil, enabling these tissues to expand and contract up to two billion times over a person’s lifetime. The two most abundant components of elastic fibres are tropoelastin and fibrillin. The process by which elastic fibres assemble is not well understood. This information is needed in regenerative medicine, which aims to replace or regenerate cells, tissues or organs to restore or establish normal function. We know that the enzyme transglutaminase-2 covalently links tropoelastin and fibrillin and that this interaction between tropoelastin and fibrillin enhances tropoelastin assembly. However, we do not understand the importance of the linkage between tropoelastin and fibrillin and why this supports elastic fibre assembly. As part of a long-term collaboration, researchers from the University of Manchester, the University of Sydney and the University of Connecticut used the BioSAXS beamline B21 to collect Small-Angle X-ray Scattering (SAXS) data. They used the data to generate models of tropoelastin and fibrillin, and a tropoelastin-fibrillin complex. These models were used to understand what effect cross-linking had on the dynamics of these proteins. The models suggest that tropoelastin and fibrillin interact in an end-to-end manner and that cross-linking these two proteins together reduces their molecular motions, suggesting a stabilising effect due to this interaction. These findings suggest that the cross-link formation between tropoelastin and fibrillin stabilises the elastin precursor so that it is primed for elastic fibre assembly.	Jul 2021
B21-High Throughput SAXS	Transglutaminase-mediated cross-linking of tropoelastin to fibrillin stabilises the elastin precursor prior to elastic fibre assembly Michael P. Lockhart-Cairns , Helena Newandee , Jennifer Thomson , Anthony S. Weiss , Clair Baldock , Anna Tarakanova Journal Of Molecular Biology DOI: 10.1016/j.jmb.2020.08.023 Diamond Proposal Number(s): [17773] Show Abstract ▼ Abstract: Elastic fibres are essential components of all mammalian elastic tissues such as blood vessels, lung and skin, and are critically important for the mechanical properties they endow. The main components of elastic fibres are elastin and fibrillin, where correct formation of elastic fibres requires a fibrillin microfibril scaffold for the deposition of elastin. It has been demonstrated previously that the interaction between fibrillin and tropoelastin, the elastin precursor, increases the rate of assembly of tropoelastin. Furthermore, tropoelastin and fibrillin can be cross-linked by transglutaminase-2 but the function of cross-linking on their elastic properties is yet to be elucidated. Here we show that transglutaminase cross-linking supports formation of a 1:1 stoichiometric fibrillin-tropoelastin complex. SAXS data show that the complex retains features of the individual proteins, but is elongated supporting end-to-end assembly. Elastic network models were constructed to compare the dynamics of tropoelastin and fibrillin individually as well as in the cross-linked complex. Normal mode analysis was performed to determine the structures' most energetically favourable, biologically accessible motions which show that within the complex, tropoelastin is less mobile and this molecular stabilisation extends along the length of the tropoelastin molecule to regions remote from the cross-linking site. Together, these data suggest a long-range stabilising effect of cross-linking that occurs due to the covalent linkage of fibrillin to tropoelastin. This work provides insight into the interactions of tropoelastin and fibrillin and how cross-link formation stabilises the elastin precursor so it is primed for elastic fibre assembly.	Sep 2020
Krios I-Titan Krios I at Diamond	Dual role of the active site ‘lid’ regions of protochlorophyllide oxidoreductase in photocatalysis and plant development Shaowei Zhang , Alan R. F. Godwin , Aoife Taylor , Samantha J. O. Hardman , Thomas A. Jowitt , Linus O. Johannissen , Sam Hay , Clair Baldock , Derren J. Heyes , Nigel S. Scrutton The Febs Journal 288, 175 - 189 DOI: 10.1111/febs.15542 Diamond Proposal Number(s): [22724] Open Access Show Abstract ▼ Abstract: Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light‐dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR–Pchlide–NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a ‘lid’ to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR ‘octamer’ has been isolated and its structure investigated by cryo‐electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation – a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.	Aug 2020
B21-High Throughput SAXS I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	The dual PDZ domain from Postsynaptic density protein 95 forms a scaffold with peptide ligand N. A. Rodzli , M. P. Lockhart-Cairns , C. W. Levy , J. Chipperfield , L. Bird , C. Baldock , S. M. Prince Biophysical Journal DOI: 10.1016/j.bpj.2020.06.018 Open Access Show Abstract ▼ Abstract: PSD-95 is a member of Membrane Associated Guanylate Kinase class of proteins which form scaffolding interactions with partner proteins including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/Disks Large/Zona Occludens (PDZ) domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of this dual domain in both in apo- and ligand-bound form; thermodynamic analysis of ligand association and Small Angle X-ray Scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a 3-dimensional scaffold which can be described by a Spacegroup. The concentration of the components in this study is comparable to those found in compartments of excitable cells such as the postsynaptic density and juxta-paranodes of Ranvier. These in vitro experiments inform the basis of the scaffolding function of PSD-95 and provide a detailed model for scaffold formation by the PDZ domains of PSD-95.	Jun 2020
I03-Macromolecular Crystallography I22-Small angle scattering & Diffraction	Inter-α-inhibitor heavy chain-1 has an integrin-like 3D structure mediating immune regulatory activities and matrix stabilization during ovulation Jan J. Enghild , Clair Baldock , Caroline M. Milner , Anthony J. Day , David C. Briggs , Alexander W. W. Langford-Smith , Holly L. Birchenough , Thomas A. Jowitt , Cay M. Kielty Journal Of Biological Chemistry DOI: 10.1074/jbc.RA119.011916 Open Access Show Abstract ▼ Abstract: Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering–based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.	Apr 2020
B21-High Throughput SAXS Krios I-Titan Krios I at Diamond	The C-terminal dimerization domain of the respiratory mucin MUC5B functions in mucin stability and intracellular packaging before secretion Caroline Ridley , Michael P. Lockhart-Cairns , Richard F. Collins , Thomas A. Jowitt , Durai B. Subramani , Mehmet Kesimer , Clair Baldock , David J. Thornton Journal Of Biological Chemistry 294, 17105 - 17116 DOI: 10.1074/jbc.RA119.010771 Diamond Proposal Number(s): [16619, 17773] Open Access Show Abstract ▼ Abstract: Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain. Here, using cryogenic electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS) analyses of recombinant disulfide-linked dimeric MUC5B dimerization domain we identified an asymmetric, elongated twisted structure, with a double globular base. We found that the dimerization domain is more resistant to disruption than the multimerization domain suggesting the twisted structure of the dimerization domain confers additional stability to MUC5B polymers. Size-exclusion chromatography-multiangle light scattering (SEC-MALS), SPR-based biophysical analyses and microscale thermophoresis of the dimerization domain disclosed no further assembly, but did reveal reversible, calcium-dependent interactions between the dimerization and multimerization domains that were most active at acidic pH, suggesting that these domains have a role in MUC5B intragranular organization. In summary, our results suggest a role for the C-terminal dimerization domain of MUC5B in compaction of mucin chains during granular packaging via interactions with the N-terminal multimerization domain. Our findings further suggest that the less stable multimerization domain provides a potential target for mucin depolymerization to remove mucus plugs in COPD and other lung pathologies.	Nov 2019
B21-High Throughput SAXS	Lysyl oxidase–like 2 (LOXL2)–mediated cross-linking of tropoelastin Christian E. H. Schmelzer , Andrea Heinz , Helen Troilo , Michael P. Lockhart-Cairns , Thomas A. Jowitt , Marion F. Marchand , Laurent Bidault , Marine Bignon , Tobias Hedtke , Alain Barret , James C. Mcconnell , Michael J. Sherratt , Stéphane Germain , David J. S. Hulmes , Clair Baldock , Laurent Muller The Faseb Journal DOI: 10.1096/fj.201801860RR Diamond Proposal Number(s): [1783] Open Access Show Abstract ▼ Abstract: Lysyl oxidases (LOXs) play a central role in extracellular matrix remodeling during development and tumor growth and fibrosis through cross-linking of collagens and elastin. We have limited knowledge of the structure and substrate specificity of these secreted enzymes. LOXs share a conserved C-terminal catalytic domain but differ in their N-terminal region, which is composed of 4 repeats of scavenger receptor cysteine-rich (SRCR) domains in LOX-like (LOXL) 2. We investigated by X-ray scattering and electron microscopy the low-resolution structure of the full-length enzyme and the structure of a shorter form lacking the catalytic domain. Our data demonstrate that LOXL2 has a rod-like structure with a stalk composed of the SRCR domains and the catalytic domain at its tip. We detected direct interaction between LOXL2 and tropoelastin (TE) and also LOXL2-mediated deamination of TE. Using proteomics, we identified several allysines together with cross-linked TE peptides. The elastin-like material generated was resistant to trypsin proteolysis and displayed mechanical properties similar to mature elastin. Finally, we detected the codistribution of LOXL2 and elastin in the vascular wall. Altogether, these data suggest that LOXL2 could participate in elastogenesis in vivo and could be used as a means of cross-linking TE in vitro for biomimetic and cell-compatible tissue engineering purposes.	Jan 2019

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