B21-High Throughput SAXS
|
Diamond Proposal Number(s):
[14891]
Open Access
Abstract: Calbindin-D28k is a calcium binding protein that is highly expressed in the mammalian central nervous system. It has been reported that calbindin-D28k binds to and increases the activity of inositol Monophosphatase (IMPase). This is an enzyme that is involved in the homeostasis of the Inositol trisphosphate signalling cascade by catalysing the final dephosphorylation of inositol and has been implicated in the therapeutic mechanism of lithium treatment of bipolar disorder. Previously studies have shown that calbindin-D28k can increase IMPase activity by up to 250 hundred-fold. A preliminary in silico model was proposed for the interaction.
Here, we aimed at exploring the shape and properties of the calbindin-IMPase complex to gain new insights on this biologically important interaction. We created several fusion constructs of calbindin-D28k and IMPase, connected by flexible amino acid linkers of different lengths and orientations to fuse the termini of the two proteins together. The resulting fusion proteins have activities 200%–400% higher the isolated wild-type IMPase. The constructs were characterized by small angle X-ray scattering to gain information on the overall shape of the complexes and validate the previous model. The fusion proteins form a V-shaped, elongated and less compact complex as compared to the model. Our results shed new light into this protein-protein interaction.
|
Jul 2022
|
|
I03-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[18666]
Open Access
Abstract: Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is poorly understood. Arc has an N-terminal domain (NTD) involved in membrane binding and a C-terminal domain (CTD) that binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerisation, including assembly of retrovirus-like capsids involved in intercellular signalling. To obtain new tools for studies on Arc structure and function, we produced and characterised six high-affinity anti-Arc nanobodies (Nb). The CTD of rat and human Arc were both crystallised in ternary complexes with two Nbs. One Nb bound deep into the stargazin-binding pocket of Arc CTD and suggested competitive binding with Arc ligand peptides. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. The collapsed conformation closely resembles Drosophila Arc in capsids, suggesting that we have trapped a capsid-like conformation of the human Arc CTD. Our data obtained with the help of anti-Arc Nbs suggest that structural dynamics of the CTD and dimerisation of the NTD may promote the formation of capsids. Taken together, the recombinant high-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function, as well as mechanisms of Arc capsid formation, both in vitro and in vivo. For example, the Nbs could serve as a genetically encoded tools for inhibition of endogenous Arc interactions in the study of neuronal function and plasticity.
|
Jun 2022
|
|
Krios I-Titan Krios I at Diamond
|
Diamond Proposal Number(s):
[20287]
Open Access
Abstract: Although amyloid fibres are highly stable protein aggregates, a specific combination of human Hsp70 system chaperones can disassemble them, including fibres formed of α-synuclein, huntingtin, or Tau. Disaggregation requires the ATPase activity of the constitutively expressed Hsp70 family member, Hsc70, together with the J domain protein DNAJB1 and the nucleotide exchange factor Apg2. Clustering of Hsc70 on the fibrils appears to be necessary for disassembly. Here we use atomic force microscopy to show that segments of in vitro assembled α-synuclein fibrils are first coated with chaperones and then undergo bursts of rapid, unidirectional disassembly. Cryo-electron tomography and total internal reflection fluorescence microscopy reveal fibrils with regions of densely bound chaperones, preferentially at one end of the fibre. Sub-stoichiometric amounts of Apg2 relative to Hsc70 dramatically increase recruitment of Hsc70 to the fibres, creating localised active zones that then undergo rapid disassembly at a rate of ~ 4 subunits per second. The observed unidirectional bursts of Hsc70 loading and unravelling may be explained by differences between the two ends of the polar fibre structure.
|
Jun 2022
|
|
B21-High Throughput SAXS
|
Diamond Proposal Number(s):
[30822]
Open Access
Abstract: Botulinum Neurotoxins (BoNT) are the most potent toxins currently known. However, they also have therapeutic applications for an increasing number of motor related conditions due to their specificity, and low diffusion into the system. Although the start- and end- points for the BoNT mechanism of action are well-studied, a critical step remains poorly understood. It is theorised that BoNTs undergo a pH-triggered conformational shift, activating the neurotoxin by priming it to form a transmembrane (TM) channel. To test this hypothesis, we combined molecular dynamic (MD) simulations and small-angle x-ray scattering (SAXS), revealing a new conformation of BoNT/E. This conformation was exclusively observed in simulations below pH 5.5, as determined by principal component analysis (PCA), and its theoretical SAXS profile matched an experimental SAXS profile obtained at pH 4. Additionally, a localised secondary structural change was observed in MD simulations below pH 5.5, in a region previously identified as instrumental for membrane insertion for BoNT/A. These changes were found at a critical pH value for BoNTs in vivo, and may be relevant for their therapeutic use.
|
Jun 2022
|
|
I13-2-Diamond Manchester Imaging
|
Diamond Proposal Number(s):
[20274]
Open Access
Abstract: Integrating physiology with structural insights of the same neuronal circuit provides a unique approach to understanding how the mammalian brain computes information. However, combining the techniques that provide both streams of data represents an experimental challenge. When studying glomerular column circuits in the mouse olfactory bulb, this approach involves e.g., recording the neuronal activity with in vivo 2-photon (2P) calcium imaging, retrieving the circuit structure with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT) and/or serial block-face scanning electron microscopy (SBEM) and correlating these datasets. Sample preparation and dataset correlation are two key bottlenecks in this correlative workflow. Here, we first quantify the occurrence of different artefacts when staining tissue slices with heavy metals to generate X-ray or electron contrast. We report improvements in the staining procedure, ultimately achieving perfect staining in ∼67% of the 0.6 mm thick olfactory bulb slices that were previously imaged in vivo with 2P. Secondly, we characterise the accuracy of the spatial correlation between functional and structural datasets. We demonstrate that direct, single-cell precise correlation between in vivo 2P and SXRT tissue volumes is possible and as reliable as correlating between 2P and SBEM. Altogether, these results pave the way for experiments that require retrieving physiology, circuit structure and synaptic signatures in targeted regions. These correlative function-structure studies will bring a more complete understanding of mammalian olfactory processing across spatial scales and time.
|
Jun 2022
|
|
I03-Macromolecular Crystallography
|
Nikolaos
Georgakopoulos
,
Sandeep
Talapatra
,
Dina
Dikovskaya
,
Sharadha
Dayalan Naidu
,
Maureen
Higgins
,
Jemma
Gatliff
,
Aysel
Ayhan
,
Roxani
Nikoloudaki
,
Marjolein
Schaap
,
Klara
Valko
,
Farideh
Javid
,
Albena T.
Dinkova-Kostova
,
Frank
Kozielski
,
Geoffrey
Wells
Diamond Proposal Number(s):
[12305]
Open Access
Abstract: Inhibitors of Kelch-like ECH-associated protein 1 (Keap1) increase the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) by stalling its ubiquitination and degradation. This enhances the expression of genes encoding proteins involved in drug detoxification, redox homeostasis, and mitochondrial function. Nrf2 activation offers a potential therapeutic approach for conditions including Alzheimer’s and Parkinson’s diseases, vascular inflammation, and chronic obstructive airway disease. Non-electrophilic Keap1-Nrf2 protein–protein interaction (PPI) inhibitors may have improved toxicity profiles and different pharmacological properties to cysteine-reactive electrophilic inhibitors. Here, we describe and characterize a series of phenyl bis-sulfonamide PPI inhibitors that bind to Keap1 at submicromolar concentrations. Structural studies reveal that the compounds bind to Keap1 in a distinct “peptidomimetic” conformation that resembles the Keap1-Nrf2 ETGE peptide complex. This is different to other small molecule Keap1-Nrf2 PPI inhibitors, including bicyclic aryl bis-sulfonamides, offering a starting point for new design approaches to Keap1 inhibitors.
|
May 2022
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
|
Nicky J.
Willis
,
William
Mahy
,
James
Sipthorp
,
Yuguang
Zhao
,
Hannah L.
Woodward
,
Benjamin N.
Atkinson
,
Elliott D.
Bayle
,
Fredrik
Svensson
,
Sarah
Frew
,
Fiona
Jeganathan
,
Amy
Monaghan
,
Stefano
Benvegnù
,
Sarah
Jolly
,
Luca
Vecchia
,
Reinis R.
Ruza
,
Svend
Kjær
,
Steven
Howell
,
Ambrosius P.
Snijders
,
Magda
Bictash
,
Patricia C.
Salinas
,
Jean-Paul
Vincent
,
E. Yvonne
Jones
,
Paul
Whiting
,
Paul V.
Fish
Diamond Proposal Number(s):
[16814, 19446]
Open Access
Abstract: Notum is a carboxylesterase that suppresses Wnt signaling through deacylation of an essential palmitoleate group on Wnt proteins. There is a growing understanding of the role Notum plays in human diseases such as colorectal cancer and Alzheimer’s disease, supporting the need to discover improved inhibitors, especially for use in models of neurodegeneration. Here, we have described the discovery and profile of 8l (ARUK3001185) as a potent, selective, and brain-penetrant inhibitor of Notum activity suitable for oral dosing in rodent models of disease. Crystallographic fragment screening of the Diamond-SGC Poised Library for binding to Notum, supported by a biochemical enzyme assay to rank inhibition activity, identified 6a and 6b as a pair of outstanding hits. Fragment development of 6 delivered 8l that restored Wnt signaling in the presence of Notum in a cell-based reporter assay. Assessment in pharmacology screens showed 8l to be selective against serine hydrolases, kinases, and drug targets.
|
May 2022
|
|
I04-Macromolecular Crystallography
|
Diamond Proposal Number(s):
[23269]
Open Access
Abstract: Botulinum neurotoxins (BoNT) are a group of clostridial toxins that cause the potentially fatal neuroparalytic disease botulism. Although highly toxic, BoNTs are utilized as therapeutics to treat a range of neuromuscular conditions. Several serotypes (BoNT/A-/G, /X) have been identified with vastly differing toxicological profiles. Each serotype can be further sub-categorised into subtypes due to subtle variations in their protein sequence. These minor changes have been attributed to differences in both the duration of action and potency for BoNT/A subtypes. BoNTs are composed of three domains—a cell-binding domain, a translocation domain, and a catalytic domain. In this paper, we present the crystal structures of the botulinum neurotoxin A2 cell binding domain, both alone and in complex with its receptor ganglioside GD1a at 1.63 and 2.10 Å, respectively. The analysis of these structures reveals a potential redox-dependent Lys-O-Cys bridge close to the ganglioside binding site and a hinge motion between the HCN and HCC subdomains. Furthermore, we make a detailed comparison with the previously reported HC/A2:SV2C structure for a comprehensive structural analysis of HC/A2 receptor binding.
|
May 2022
|
|
I04-1-Macromolecular Crystallography (fixed wavelength)
I24-Microfocus Macromolecular Crystallography
|
Open Access
Abstract: UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2′s third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B’s disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3′s middle-domain elucidates its essential role in NMD.
|
May 2022
|
|
I13-2-Diamond Manchester Imaging
|
Carles
Bosch
,
Tobias
Ackels
,
Alexandra
Pacureanu
,
Yuxin
Zhang
,
Christopher J.
Peddie
,
Manuel
Berning
,
Norman
Rzepka
,
Marie-Christine
Zdora
,
Isabell
Whiteley
,
Malte
Storm
,
Anne
Bonnin
,
Christoph
Rau
,
Troy
Margrie
,
Lucy
Collinson
,
Andreas T.
Schaefer
Diamond Proposal Number(s):
[20274]
Open Access
Abstract: Understanding the function of biological tissues requires a coordinated study of physiology and structure, exploring volumes that contain complete functional units at a detail that resolves the relevant features. Here, we introduce an approach to address this challenge: Mouse brain tissue sections containing a region where function was recorded using in vivo 2-photon calcium imaging were stained, dehydrated, resin-embedded and imaged with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT). SXRT provided context at subcellular detail, and could be followed by targeted acquisition of multiple volumes using serial block-face electron microscopy (SBEM). In the olfactory bulb, combining SXRT and SBEM enabled disambiguation of in vivo-assigned regions of interest. In the hippocampus, we found that superficial pyramidal neurons in CA1a displayed a larger density of spine apparati than deeper ones. Altogether, this approach can enable a functional and structural investigation of subcellular features in the context of cells and tissues.
|
May 2022
|
|
