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

Instruments / Technical Area	Publication Details	Published
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
B21-High Throughput SAXS	Internal cleavage and synergy with twisted gastrulation enhance BMP inhibition by BMPER Michael P. Lockhart-Cairns , Karen Tzia Wei Lim , Alexandra Zuk , Alan R. F. Godwin , Stuart A. Cain , Gerhard Sengle , Clair Baldock Matrix Biology DOI: 10.1016/j.matbio.2018.08.006 Diamond Proposal Number(s): [1580] Open Access Show Abstract ▼ Abstract: Bone morphogenetic proteins (BMPs) are essential signalling molecules involved in developmental and pathological processes and are regulated in the matrix by secreted glycoproteins. One such regulator is BMP-binding endothelial cell precursor-derived regulator (BMPER) which can both inhibit and enhance BMP signalling in a context and concentration-dependent manner. Twisted gastrulation (Tsg) can also promote or ablate BMP activity but it is unclear whether Tsg and BMPER directly interact and thereby exert a synergistic function on BMP signalling. Here, we show that human BMPER binds to Tsg through the N-terminal BMP-binding region which alone more potently inhibits BMP-4 signalling than full-length BMPER. Additionally, BMPER and Tsg cooperatively inhibit BMP-4 signalling suggesting a synergistic function to dampen BMP activity. Furthermore, full-length BMPER is targeted to the plasma membrane via binding of its C-terminal region to cell surface heparan sulphate proteoglycans but the active cleavage fragment is diffusible. Small-angle X-ray scattering and electron microscopy show that BMPER has an elongated conformation allowing the N-terminal BMP-binding and C-terminal cell-interactive regions to be spatially separated. To gain insight into the regulation of BMPER bioavailability by internal cleavage, a disease-causing BMPER point mutation, P370L, previously identified in the acid-catalysed cleavage site, was introduced. The mutated protein was secreted but the mutation prevented intracellular cleavage resulting in a lack of bioactive cleavage fragment. Furthermore, mutant BMPER was extracellularly cleaved at a downstream site presumably becoming available due to the mutation. This susceptibility to extracellular proteases and loss of bioactive N-terminal cleavage fragment may result in loss of BMPER function in disease.	Aug 2018
B21-High Throughput SAXS	Coarse-grained modeling of antibodies from small-angle scattering profiles Daniel Corbett , Max Hebditch , Rose Keeling , Peng Ke , Sofia Ekizoglou , Prasad Sarangapani , Jai Pathak , Christopher F. Van Der Walle , Shahid Uddin , Clair Baldock , Carlos Avendaño , Robin A. Curtis The Journal Of Physical Chemistry B 121, 8276 - 8290 DOI: 10.1021/acs.jpcb.7b04621 Diamond Proposal Number(s): [10510] Show Abstract ▼ Abstract: Predicting the concentrated solution behavior for monoclonal antibodies requires developing and using minimal models to describe their shape and interaction potential. Toward this end, the small-angle X-ray scattering (SAXS) profiles for a monoclonal antibody (COE-03) have been measured under solution conditions chosen to produce weak self-association. The experiments are complemented with molecular simulations of a three-bead antibody model with and without interbead attraction. The scattering profile is extracted directly from the molecular simulation to avoid using the decoupling approximation. We examine the ability of the three-bead model to capture features of the scattering profile and the dependence of compressibilty on protein concentration. The three-bead model is able to reproduce generic features of the experimental structure factor as a function of wave vector S(k) including a well-defined shoulder, which is a consequence of the planar structure of the antibody, and a well-defined minimum in S(k) at k ∼ 0.025 Å–1. We also show the decoupling approximation is incapable of accounting for highly anisotropic shapes. The best-fit parameters obtained from matching spherical models to simulated scattering profiles are protein concentration dependent, which limits their applicability for predicting thermodynamic properties. Nevertheless, the experimental compressibility curves can be accurately reproduced by an appropriate parametrization of the Baxter adhesive model, indicating the model provides a semiempirical equation of state for the antibody. The results provide insights into how equations of state can be improved for antibodies by accounting for their anisotropic shapes.	Aug 2017

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