I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[21970]
Open Access
Abstract: Oral and gut microbiomes are important for the maintenance of homeostasis in the human body. Altered or disturbed mutualism between their members results in dysbiosis with local injury and subsequent systemic diseases. The high bacterial density causes intense competition among microbiome residents to acquire nutrients, including iron and heme, the latter of high importance for heme auxotrophic members of the Bacteroidetes phylum. Our main hypothesis is that the heme acquisition mechanism, with the leading role played by a novel HmuY family of hemophore-like proteins, can be used to fulfill nutritional requirements and increase virulence. We characterized HmuY homologs expressed by Bacteroides fragilis and compared their properties with the first representative of this family, the HmuY protein of Porphyromonas gingivalis. In contrast to other Bacteroidetes members, B. fragilis produces three HmuY homologs (Bfr proteins). All bfr transcripts were produced at higher levels in bacteria starved of iron and heme (fold change increase ~60, ~90, and ~70 for bfrA, bfrB, and bfrC, respectively). X-ray protein crystallography showed that B. fragilis Bfr proteins are structurally similar to P. gingivalis HmuY and to other homologs, except for differences in the potential heme-binding pockets. BfrA binds heme, mesoheme, and deuteroheme, but preferentially under reducing conditions, using Met175 and Met146 to coordinate heme iron. BfrB binds iron-free protoporphyrin IX and coproporphyrin III, whereas BfrC does not bind porphyrins. HmuY is capable of heme sequestration from BfrA, which might increase the ability of P. gingivalis to cause dysbiosis also in the gut microbiome.
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Jul 2023
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[16557]
Open Access
Abstract: Achilles tendon rupture is a common debilitating medical condition. The healing process is slow and can be affected by heterotopic ossification (HO), which occurs when pathologic bone-like tissue is deposited instead of the soft collagenous tendon tissue. Little is known about the temporal and spatial progression of HO during Achilles tendon healing. In this study we characterize HO deposition, microstructure, and location at different stages of healing in a rat model. We use phase contrast-enhanced synchrotron microtomography, a state-of-the-art technique that allows 3D imaging at high-resolution of soft biological tissues without invasive or time-consuming sample preparation. The results increase our understanding of HO deposition, from the early inflammatory phase of tendon healing, by showing that the deposition is initiated as early as one week after injury in the distal stump and mostly growing on preinjury HO deposits. Later, more deposits form first in the stumps and then all over the tendon callus, merging into large, calcified structures, which occupy up to 10% of the tendon volume. The HOs were characterized by a looser connective trabecular-like structure and a proteoglycan-rich matrix containing chondrocyte-like cells with lacunae. The study shows the potential of 3D imaging at high-resolution by phase-contrast tomography to better understand ossification in healing tendons.
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Jun 2023
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I13-2-Diamond Manchester Imaging
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Aïcha
Ben Zemzem
,
Aline
Genevaux
,
Amandine
Wahart
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Andrew J.
Bodey
,
Sébastien
Blaise
,
Béatrice
Romier-Crouzet
,
Jessica
Jonquet
,
Camille
Bour
,
Rémi
Cogranne
,
Pierre
Beauseroy
,
Manuel
Dauchez
,
Michael J.
Sherratt
,
Laurent
Debelle
,
Sébastien
Almagro
Diamond Proposal Number(s):
[12776]
Open Access
Abstract: The arterial wall consists of three concentric layers: intima, media, and adventitia. Beyond their resident cells, these layers are characterized by an extracellular matrix (ECM), which provides both biochemical and mechanical support. Elastin, the major component of arterial ECM, is present in the medial layer and organized in concentric elastic lamellae that confer resilience to the wall. We explored the arterial wall structures from C57Bl6 (control), db/db (diabetic), and ApoE−/− (atherogenic) mice aged 3 months using synchrotron X-ray computed microtomography on fixed and unstained tissues with a large image field (8 mm3). This approach combined a good resolution (0.83 µm/voxel), large 3D imaging field. and an excellent signal to noise ratio conferred by phase-contrast imaging. We determined from 2D virtual slices that the thickness of intramural ECM structures was comparable between strains but automated image analysis of the 3D arterial volumes revealed a lattice-like network within concentric elastic lamellae. We hypothesize that this network could play a role in arterial mechanics. This work demonstrates that phase-contrast synchrotron X-ray computed microtomography is a powerful technique which to characterize unstained soft tissues.
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Oct 2021
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[20975]
Abstract: Nitrilases are commercially important biocatalysts found in diverse taxa that catalyse the hydrolysis of nitriles to amides and acids. In vitro they form rings, terminating spirals or long filaments that seem to be necessary for activity. In vivo, NIT1 assemblies have been observed in Arabidopsis. Substrate size is correlated with the helical twist of nitrilase filaments and can be altered by changing a single residue by site‐directed mutagenesis or by mixing with an excess of enzyme in a different helical conformation. We have determined the first close‐to‐atomic resolution structure of an active nitrilase by cryo‐EM and identified substrate‐specifying residues for site saturation directed evolution. In silico docking of the substrate, β‐L‐cyanoalanine, into the binding pocket led to the identification of a loop that limits the maximum length of a bound substrate and shifts position as a function of helical twist. We propose that the overall size of the binding pocket is specified by this loop, while binding pocket residues alter selectivity for different functional groups. We propose that this mechanism allows nitrilases to rapidly neofunctionalise according to cellular requirements.
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Apr 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[18069, 12346]
Open Access
Abstract: Plasmodium falciparum is the most lethal of human-infective malaria parasites. A hallmark of P. falciparum malaria is extensive remodeling of host erythrocytes by the parasite, which facilitates the development of virulence properties such as host cell adhesion to the endothelial lining of the microvasculature. Host remodeling is mediated by a large complement of parasite proteins exported to the erythrocyte; among them is a single heat shock protein (Hsp)70–class protein chaperone, P. falciparum Hsp70-x (PfHsp70-x). PfHsp70-x was previously shown to assist the development of virulent cytoadherence characteristics. Here, we show that PfHsp70-x also supports parasite growth under elevated temperature conditions that simulate febrile episodes, especially at the beginning of the parasite life cycle when most of host cell remodeling takes place. Biochemical and biophysical analyses of PfHsp70-x, including crystallographic structures of its catalytic domain and the J-domain of its stimulatory Hsp40 cochaperone, suggest that PfHsp70-x is highly similar to human Hsp70 chaperones endogenous to the erythrocyte. Nevertheless, our results indicate that selective inhibition of PfHsp70-x function using small molecules may be possible and highlight specific sites of its catalytic domain as potentially of high interest. We discuss the likely roles of PfHsp70-x and human chaperones in P. falciparum biology and how specific inhibitors may assist us in disentangling their relative contributions.
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Nov 2019
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I04-Macromolecular Crystallography
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Abstract: d‐Amino acids are the “wrong” enantiomers of amino acids as they are not used in proteins synthesis but evolved in selected functions. On this side, d‐aspartate (d‐Asp) plays several significant roles in mammals, especially as an agonist of N‐methyl‐d‐aspartate receptors (NMDAR), and is involved in relevant diseases, such as schizophrenia and Alzheimer's disease. In vivo modulation of d‐Asp levels represents an intriguing task to cope with such pathological states. As little is known about d‐Asp synthesis, the only option for modulating the levels is via degradation, which is due to the flavoenzyme d‐aspartate oxidase (DASPO). Here we present the first three‐dimensional structure of a DASPO enzyme (from human) which belongs to the d‐amino acid oxidase family. Notably, human DASPO differs from human d‐amino acid oxidase (attributed to d‐serine degradation, the main coagonist of NMDAR) showing peculiar structural features (a specific active site charge distribution), oligomeric state and kinetic mechanism, and a higher FAD affinity and activity. These results provide useful insights into the structure‐function relationships of human DASPO: modulating its activity represents now a feasible novel therapeutic target.
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Nov 2019
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Elena
Tibaldi
,
Alessandra
Brocca
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Antonietta
Sticca
,
Elisabetta
Gola
,
Marco
Pizzi
,
Luciana
Bordin
,
Mario Angelo
Pagano
,
Marco
Mazzorana
,
Gabriella
Donà
,
Paola
Violi
,
Oriano
Marin
,
Antonella
Romano
,
Paolo
Angeli
,
Amedeo
Carraro
,
Anna Maria
Brunati
Abstract: Osteopontin (OPN) is a phosphoglycoprotein secreted into the extracellular matrix upon liver injury, acting as a cytokine stimulates the deposition of fibrillary collagen in liver fibrogenesis. In livers of mice subjected to bile duct ligation (BDL) and in cultured activated hepatic stellate cells (HSCs), we show that OPN, besides being overexpressed, is substantially phosphorylated by family with sequence similarity 20, member C (Fam20C), formerly known as Golgi casein kinase (G‐CK), which is exclusively resident in the Golgi apparatus. In both experimental models, Fam20C becomes overactive when associated with a 500‐kDa multiprotein complex, as compared with the negligible activity in livers of sham‐operated rats and in quiescent HSCs. Fam20C knockdown not only confirmed the role of Fam20C itself in OPN phosphorylation, but also revealed that phosphorylation was essential for OPN secretion. However, OPN acts as a fibrogenic factor independently of its phosphorylation state, as demonstrated by the increased expression of Collagen‐I by HSCs incubated with either a phosphorylated or nonphosphorylated form of recombinant OPN. Collectively, our results confirm that OPN promotes liver fibrosis and highlight Fam20C as a novel factor driving this process by favoring OPN secretion from HSCs, opening new avenues for deciphering yet unidentified mechanisms underlying liver fibrogenesis.
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Nov 2019
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B21-High Throughput SAXS
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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
Diamond Proposal Number(s):
[1783]
Open Access
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.
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Jan 2019
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I04-Macromolecular Crystallography
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Open Access
Abstract: Uniquely among malaria parasites, Plasmodium falciparum-infected erythrocytes (iRBCs) develop membrane protrusions, known as knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed subtelomeric (PHIST) domains has attracted attention, with members being implicated in host-parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 μM, comigrates with PfEMP1 during export, and locates in knobs. PHIST variants that do not locate in knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1-cytoskeletal connection in knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome.Oberli, A., Slater, L. M., Cutts, E., Brand, F., Mundwiler-Pachlatko, E., Rusch, S., Masik, M. F. G., Erat, M. C., Beck, H.-P., Vakonakis, I. A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface.
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Sep 2014
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[6385]
Abstract: Much attention is focused on the benzoquinone ansamycins as anticancer agents, with several derivatives of the natural product geldanamycin (GdA) now in clinical trials. These drugs are selective inhibitors of Hsp90, a molecular chaperone vital for many of the activities that drive cancer progression. Mutational changes to their interaction site, the extremely conserved ATP binding site of Hsp90, would mostly be predicted to inactivate the chaperone. As a result, drug resistance should not arise readily this way. Nevertheless, Streptomyces hygroscopicus, the actinomycete that produces GdA, has evolved an Hsp90 family protein (HtpG) that lacks GdA binding. It is altered in certain of the highly conserved amino acids making contacts to this antibiotic in crystal structures of GdA bound to eukaryotic forms of Hsp90. Two of these amino acid changes, located on one side of the nucleotide-binding cleft, weakened GdA/Hsp90 binding and conferred partial GdA resistance when inserted into the endogenous Hsp90 of yeast cells. Crystal structures revealed their main effect to be a weakening of interactions with the C-12 methoxy group of the GdA ansamycin ring. This is the first study to demonstrate that partial GdA resistance is possible by mutation within the ATP binding pocket of Hsp90.—Millson, S. H., Chua, C.-S., Roe, S. M., Polier, S., Solovieva, S., Pearl, L. H., Sim, T.-S., Prodromou, C., Piper, P. W. Features of the Streptomyces hygroscopicus HtpG reveal how partial geldanamycin resistance can arise with mutation to the ATP binding pocket of a eukaryotic Hsp90.
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Jul 2011
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