I13-2-Diamond Manchester Imaging
|
Diamond Proposal Number(s):
[22346, 21697]
Open Access
Abstract: Magnesium is attractive for the application as a temporary bone implant due to its inherent biodegradability, non-toxicity and suitable mechanical properties. The degradation process of magnesium in physiological environments is complex and is thought to be a diffusion-limited transport problem. We use a multi-scale imaging approach using micro computed tomography and transmission X-ray microscopy (TXM) at resolutions below 40 nm. Thus, we are able to evaluate the nanoporosity of the degradation layer and infer its impact on the degradation process of pure magnesium in two physiological solutions. Magnesium samples were degraded in simulated body fluid (SBF) or Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) for one to four weeks. TXM reveals the three-dimensional interconnected pore network within the degradation layer for both solutions. The pore network morphology and degradation layer composition are similar for all samples. By contrast, the degradation layer thickness in samples degraded in SBF was significantly higher and more inhomogeneous than in DMEM+10%FBS. Distinct features could be observed within the degradation layer of samples degraded in SBF, suggesting the formation of microgalvanic cells, which are not present in samples degraded in DMEM+10%FBS. The results suggest that the nanoporosity of the degradation layer and the resulting ion diffusion processes therein have a limited influence on the overall degradation process. This indicates that the influence of organic components on the dampening of the degradation rate by the suppression of microgalvanic degradation is much greater in the present study.
|
Dec 2021
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Leandro
Oliveira Bortot
,
Victor
Lopes Rangel
,
Francesca A.
Pavlovici
,
Kamel
El Omari
,
Armin
Wagner
,
Jose
Brandao-Neto
,
Romain
Talon
,
Frank
Von Delft
,
Andrew G.
Reidenbach
,
Sonia M.
Vallabh
,
Eric
Vallabh Minikel
,
Stuart
Schreiber
,
Maria Cristina
Nonato
Diamond Proposal Number(s):
[18954]
Abstract: Prion disease is caused by the misfolding of the cellular prion protein, PrPC, into a self-templating conformer, PrPSc. Nuclear magnetic resonance (NMR) and X-ray crystallography revealed the 3D structure of the globular domain of PrPC and the possibility of its dimerization via an interchain disulfide bridge that forms due to domain swap or by non-covalent association of two monomers. On the contrary, PrPSc is composed by a complex and heterogeneous ensemble of poorly defined conformations and quaternary arrangements that are related to different patterns of neurotoxicity. Targeting PrPC with molecules that stabilize the native conformation of its globular domain emerged as a promising approach to develop anti-prion therapies. One of the advantages of this approach is employing structure-based drug discovery methods to PrPC. Thus, it is essential to expand our structural knowledge about PrPC as much as possible to aid such drug discovery efforts. In this work, we report a crystallographic structure of the globular domain of human PrPC that shows a novel dimeric form and a novel oligomeric arrangement. We use molecular dynamics simulations to explore its structural dynamics and stability and discuss potential implications of these new quaternary structures to the conversion process.
|
Dec 2021
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Diamond Proposal Number(s):
[20221]
Open Access
Abstract: Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2Apro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2Apro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2Apro of genetic groups A and B hinder 2Apro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2Apro cleave the substrate GAB1 at different sites, the 2Apro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2Apro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2Apro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2Apro as a potential target for broad-spectrum anti-enteroviral inhibitors.
|
Oct 2021
|
|
I13-2-Diamond Manchester Imaging
|
Aïcha
Ben Zemzem
,
Aline
Genevaux
,
Amandine
Wahart
,
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.
|
Oct 2021
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[19880]
Open Access
Abstract: Transcription factors are the primary regulators of gene expression and recognize specific DNA sequences under diverse physiological conditions. Although they are vital for many important cellular processes, it remains unclear when and how transcription factors and DNA interact. The antitoxin from a toxin–antitoxin system is an example of negative transcriptional autoregulation: during expression of the cognate toxin it is suppressed through binding to a specific DNA sequence. In the present study, the antitoxin HigA2 from Mycobacterium tuberculosis M37Rv was structurally examined. The crystal structure of M. tuberculosis HigA2 comprises three sections: an N-terminal autocleavage region, an α-helix bundle which contains an HTH motif, and a C-terminal β-lid. The N-terminal region is responsible for toxin binding, but was shown to cleave spontaneously in its absence. The HTH motif performs a key role in DNA binding, with the C-terminal β-lid influencing the interaction by mediating the distance between the motifs. However, M. tuberculosis HigA2 exhibits a unique coordination of the HTH motif and no DNA-binding activity is detected. Three crystal structures of M. tuberculosis HigA2 show a flexible alignment of the HTH motif, which implies that the motif undergoes structural rearrangement to interact with DNA. This study reveals the molecular mechanisms of how transcription factors interact with partner proteins or DNA.
|
Sep 2021
|
|
B21-High Throughput SAXS
|
Diamond Proposal Number(s):
[14794]
Open Access
Abstract: Mycobacterium tuberculosis (Mtb), which is responsible for more than a million deaths annually, uses lipids as the source of carbon and energy for its survival in the latent phase of infection. Mtb cannot synthesize all of the lipid molecules required for its growth and pathogenicity. Therefore, it relies on transporters such as the mammalian cell entry (Mce) complexes to import lipids from the host across the cell wall. Despite their importance for the survival and pathogenicity of Mtb, information on the structural properties of these proteins is not yet available. Each of the four Mce complexes in Mtb (Mce1–4) comprises six substrate-binding proteins (SBPs; MceA–F), each of which contains four conserved domains (N-terminal transmembrane, MCE, helical and C-terminal unstructured tail domains). Here, the properties of the various domains of Mtb Mce1A and Mce4A, which are involved in the import of mycolic/fatty acids and cholesterol, respectively, are reported. In the crystal structure of the MCE domain of Mce4A (MtMce4A39–140) a domain-swapped conformation is observed, whereas solution studies, including small-angle X-ray scattering (SAXS), indicate that all Mce1A and Mce4A domains are predominantly monomeric. Further, structural comparisons show interesting differences from the bacterial homologs MlaD, PqiB and LetB, which form homohexamers when assembled as functional transporter complexes. These data, and the fact that there are six SBPs in each Mtb mce operon, suggest that the MceA–F SBPs from Mce1–4 may form heterohexamers. Also, interestingly, the purification and SAXS analysis showed that the helical domains interact with the detergent micelle, suggesting that when assembled the helical domains of MceA–F may form a hydrophobic pore for lipid transport, as observed in EcPqiB. Overall, these data highlight the unique structural properties of the Mtb Mce SBPs.
|
Sep 2021
|
|
I02-Macromolecular Crystallography
I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Diamond Proposal Number(s):
[19951, 26302, 14794, 10291]
Open Access
Abstract: The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2β2 tetrameric enzyme. The α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) activities and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) activity. Enzyme kinetic data reported here show that medium and long chain enoyl-CoA molecules are preferred substrates for MtTFE. Modelling studies indicate how the linear medium and long acyl chains of these substrates can bind to each of the active sites. In addition, crystallographic binding studies have identified three new CoA binding sites which are different from the previously known CoA binding sites of the three TFE active sites. Structure comparisons provide new insights into the properties of ECH, HAD and KAT active sites of MtTFE. The interactions of the adenine moiety of CoA with loop-2 of the ECH active site cause a conformational change of this loop by which a competent ECH active site is formed. The NAD+ binding domain (domain C) of the HAD part of MtTFE has only a few interactions with the rest of the complex and adopts a range of open conformations, whereas the A-domain of the ECH part is rigidly fixed with respect to the HAD part. Two loops, the CB1-CA1 region and the catalytic CB4-CB5 loop, near the thiolase active site and the thiolase dimer interface, have high B-factors. Structure comparisons suggest that a competent and stable thiolase dimer is formed only when complexed with the α-chains, highlighting the importance of the assembly for the proper functioning of the complex.
|
Sep 2021
|
|
I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
|
Gustavo
Fenalti
,
Nicolas
Villanueva
,
Mark
Griffith
,
Barbra
Pagarigan
,
Sirish Kaushik
Lakkaraju
,
Richard Y.-C.
Huang
,
Nadia
Ladygina
,
Alok
Sharma
,
David
Mikolon
,
Mahan
Abbasian
,
Jeffrey
Johnson
,
Haralambos
Hadjivassiliou
,
Dan
Zhu
,
Philip P.
Chamberlain
,
Ho
Cho
,
Kandasamy
Hariharan
Open Access
Abstract: CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47’s ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention.
|
Sep 2021
|
|
B21-High Throughput SAXS
|
Diamond Proposal Number(s):
[13550]
Open Access
Abstract: Lymphostatin (LifA) is a 366 kDa protein expressed by attaching & effacing Escherichia coli. It plays an
important role in intestinal colonisation and inhibits the mitogen- and antigen-stimulated proliferation of
lymphocytes and the synthesis of proinflammatory cytokines. LifA exhibits N-terminal homology with
the glycosyltransferase domain of large clostridial toxins (LCTs). A DTD motif within this region is required
for lymphostatin activity and binding of the sugar donor uridine diphosphate N-acetylglucosamine. As with
LCTs, LifA also contains a cysteine protease motif (C1480, H1581, D1596) that is widely conserved within
the YopT-like superfamily of cysteine proteases. By analogy with LCTs, we hypothesised that the CHD
motif may be required for intracellular processing of the protein to release the catalytic N-terminal domain
after uptake and low pH-stimulated membrane insertion of LifA within endosomes. Here, we created and
validated a C1480A substitution mutant in LifA from enteropathogenic E. coli strain E2348/69. The purified
protein was structurally near-identical to the wild-type protein. In bovine T lymphocytes treated with wildtype
LifA, a putative cleavage product of approximately 140 kDa was detected. Appearance of the putative
cleavage product was inhibited in a concentration-dependent manner by bafilomycin A1 and chloroquine,
which inhibit endosome acidification. The cleavage product was not observed in cells treated with the
C1480A mutant of LifA. Lymphocyte inhibitory activity of the purified C1480A protein was significantly
impaired. The data indicate that an intact cysteine protease motif is required for cleavage of lymphostatin
and its activity against T cells.
|
Sep 2021
|
|
B21-High Throughput SAXS
|
Open Access
Abstract: Lipid cubic phase (LCP) formulations enhance the intestinal solubility and bioavailability of hydrophobic drugs by reducing precipitation and facilitating their mass transport to the intestinal surface for absorption. LCPs with an ester linkage connecting the acyl chain to the glycerol backbone (monoacylglycerols), are susceptible to chemical digestion by several lipolytic enzymes including lipases, accelerating the release of hydrophobic agents from the lipid bilayers of the matrix. Unlike regular enzymes that transform soluble substrates, lipolytic enzymes act at the interface of water and insoluble lipid. Therefore, compounds that bind to this interface can enhance or inhibit the activity of enzymes to varying extent. Here, we explore how the lipolysis rate can be tuned by the interfacial interaction of porcine pancreatic lipase with monoolein LCPs containing a known lipase inhibitor, tetrahydrolipstatin. Release of the Biopharmaceutical Classification System (BCS) class IV drug, paclitaxel, from the inhibitor-modified LCP was examined in the presence of lipase and its effectors colipase and calcium. By combining experimental dynamic digestion studies, thermodynamic measurements and molecular dynamics simulations of the competitive inhibition of lipase by tetrahydrolipstatin, we reveal the role and mode of action of lipase effectors in creating a precisely-balanced degradation-controlled LCP release system for the poorly soluble paclitaxel drug.
|
Sep 2021
|
|
