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

Instruments / Technical Area	Publication Details	Published
I04-Macromolecular Crystallography	Insights from the crystal structure of the chicken CREB3 bZIP suggest members of the CREB3 subfamily transcription factors may be activated in response to oxidative stress Keshalini Sabaratnam , Max Renner , Guido Paesen , Karl Harlos , Venugopal Nair , Raymond J. Owens , Jonathan M. Grimes Protein Science DOI: 10.1002/pro.3573 Diamond Proposal Number(s): [14744] Show Abstract ▼ Abstract: cAMP response element binding protein 3 (CREB3) is an endoplasmic reticulum (ER) membrane‐bound transcription factor, which belongs to the basic leucine zipper (bZIP) superfamily of eukaryotic transcription factors. CREB3 plays a role in the ER‐stress induced unfolded protein response (UPR) and is a multifunctional cellular factor implicated in a number of biological processes including cell proliferation and migration, tumour suppression, and immune‐related gene expression. To gain structural insights into the transcription factor, we determined the crystal structure of the conserved bZIP domain of chicken CREB3 (chCREB3) to a resolution of 3.95Å. The X‐ray structure provides evidence that chCREB3 can form a stable homodimer. The chCREB3 bZIP has a structured, pre‐formed DNA binding region, even in the absence of DNA, a feature that could potentially enhance both the DNA binding specificity and affinity of chCREB3. Significantly, the homodimeric bZIP possesses an intermolecular disulphide bond that connects equivalent cysteine residues of the parallel helices in the leucine zipper region. This disulphide bond in the hydrophobic core of the bZIP may increase the stability of the homodimer under oxidising conditions. Moreover, sequence alignment of bZIP sequences from chicken, human and mouse reveals only members of the CREB3 subfamily contain this cysteine residue, indicating it could act as a redox‐sensor. Taken together, these results suggest activity of these transcription factors may be redox‐regulated and they may be activated in response to oxidative stress.	Jan 2019
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	A new structural class of bacterial thioester domains reveals a slipknot topology Ona K. Miller , Mark J. Banfield , Ulrich Schwarz-linek Protein Science DOI: 10.1002/pro.3478 Diamond Proposal Number(s): [10071, 14980] Show Abstract ▼ Abstract: An increasing number of surface‐associated proteins identified in Gram‐positive bacteria are characterized by intramolecular cross‐links in structurally conserved thioester, isopeptide, and ester domains (TIE proteins). Two classes of thioester domains (TEDs) have been predicted based on sequence with, to date, only representatives of class I structurally characterized. Here, we present crystal structures of three class II TEDs from Bacillus anthracis, vancomycin‐resistant Staphylococcus aureus, and vancomycin‐resistant Enterococcus faecium. These proteins are structurally distinct from class I TEDs due to a β‐sandwich domain that is inserted into the conserved TED fold to form a slipknot structure. Further, the B. anthracis TED domain is presented in the context of a full‐length sortase‐anchored protein structure (BaTIE). This provides insight into the three‐dimensional arrangement of TIE proteins, which emerge as very abundant putative adhesins of Gram‐positive bacteria.	Jul 2018
I04-Macromolecular Crystallography	Redox regulated methionine oxidation of Arabidopsis thaliana glutathione transferase Phi9 induces H-site flexibility Maria-armineh Tossounian , Khadija Wahni , Inge Van Molle , Didier Vertommen , Leonardo Astolfi Rosado , Joris Messens Protein Science DOI: 10.1002/pro.3440 Diamond Proposal Number(s): [9426] Show Abstract ▼ Abstract: Glutathione transferase enzymes help plants to cope with biotic and abiotic stress. They mainly catalyse the conjugation of glutathione (GSH) onto xenobiotics, and some act as glutathione peroxidase. With X‐ray crystallography, kinetics and thermodynamics, we studied the impact of oxidation on Arabidopsis thaliana glutathione transferase Phi 9 (GSTF9). GSTF9 has no cysteine in its sequence, and it adopts a universal GST structural fold characterized by a typical conserved GSH‐binding site (G‐site) and a hydrophobic co‐substrate‐binding site (H‐site). At elevated H2O2 concentrations, methionine sulfur oxidation decreases its transferase activity. This oxidation increases the flexibility of the H‐site loop, which is reflected in lower activities for hydrophobic substrates. Determination of the transition state thermodynamic parameters shows that upon oxidation an increased enthalpic penalty is counterbalanced by a more favourable entropic contribution. All in all, to guarantee functionality under oxidative stress conditions, GSTF9 employs a thermodynamic and structural compensatory mechanism and becomes substrate of methionine sulfoxide reductases, making it a redox regulated enzyme.	May 2018
I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	X-ray crystal structures of the type IVb secretion system DotB ATPases Marie Prevost , Gabriel Waksman Protein Science DOI: 10.1002/pro.3439 Show Abstract ▼ Abstract: Human infections by the intracellular bacterial pathogen Legionella pneumophila result in a severe form of pneumonia, the Legionnaire's disease. L. pneumophila utilises a type IVb secretion (T4bS) system termed “dot/icm” to secrete protein effectors to the host cytoplasm. The dot/icm system is powered at least in part by a functionally critical AAA+ ATPase, a protein called DotB, thought to belong to the VirB11 family of proteins. Here we present the crystal structure of DotB at 3.19 Å resolution, in its hexameric form. We observe that DotB is in fact a structural intermediate between VirB11 and PilT family proteins, with a PAS‐like N‐terminal domain coupled to a RecA‐like C‐terminal domain. It also shares critical structural elements only found in PilT. The structure also reveals two conformers, termed α and β, with an αβαβαβ configuration. The existence of α and β conformers in this class of proteins was confirmed by solving the structure of DotB from another bacterial pathogen, Yersinia, where, intriguingly, we observed an ααβααβ configuration. The two conformers co‐exist regardless of the nucleotide‐bound states of the proteins. Our investigation therefore reveals that these ATPases can adopt a wider range of conformational states than was known before, shedding new light on the extraordinary spectrum of conformations these ATPases can access to carry out their function. Overall, the structure of DotB provides a template for further rational drug‐design to develop more specific antibiotics to tackle Legionnaire's disease.	May 2018
I04-Macromolecular Crystallography	Structure of an acetylating aldehyde dehydrogenase from the thermophilic ethanologen Geobacillus thermoglucosidasius Jonathan Extance , Michael J. Danson , Susan J. Crennell Protein Science DOI: 10.1002/pro.3027 Diamond Proposal Number(s): [7131] Show Abstract ▼ Abstract: Acetylating aldehyde dehydrogenases (AcAldDH) catalyse the acetylation of Coenzyme-A (CoA), or in reverse generate acetaldehyde from Acetyl-CoA using NADH as a co-factor. This paper reports the expression, purification, enzyme assay and X-ray crystal structures of an AcAldDH from Geobacillus thermoglucosidasius (GtAcAldDH) to 2.1Å and in complex with CoA and NAD(+) to 4.0Å. In the structure, the AcAldDH forms a close-knit dimer, similar to that seen in other Alcohol Dehydrogenase (ADH) structures. In GtAcAldDH these dimers associate via their N-termini to form weakly-interacting tetramers. This mode of tetrameric association is also seen in an unpublished AcAldDH deposited in the PDB, but is in contrast to all other ADH structures, (including the one other published AcAldDH found in a bacterial microcompartment), in which the dimers bury a large surface area including the C-termini. This novel mode of association sequesters the active sites and potentially-reactive acyl-enzyme intermediates in the centre of the tetramer. In other respects the structure is very similar to the other AcAldDH, binding the cofactors in a corresponding fashion. This similarity enabled the identification of a shortened substrate cavity in G. thermoglucosidasius AcAldDH, explaining the limitations on the length of substrate accepted by the enzyme.	Aug 2016
I24-Microfocus Macromolecular Crystallography	Crystal structure and analysis of HdaB: The enteroaggregative Escherichia coli AAF/IV pilus tip protein Wei-chao Lee , Steve Matthews , James A. Garnett Protein Science DOI: 10.1002/pro.2982 Diamond Proposal Number(s): [12579] Open Access Show Abstract ▼ Abstract: Enteroaggregative Escherichia coli is the primary cause of pediatric diarrhea in developing countries. They utilize aggregative adherence fimbriae (AAFs) to promote initial adherence to the host intestinal mucosa, promote the formation of biofilms, and mediate host invasion. Five AAFs have been identified to date and AAF/IV is amongst the most prevalent found in clinical isolates. Here we present the X-ray crystal structure of the AAF/IV tip protein HdaB at 2.0 Å resolution. It shares high structural homology with members of the Afa/Dr superfamily of fimbriae, which are involved in host invasion. We highlight surface exposed residues that share sequence homology and propose that these may function in invasion and also non-conserved regions that could mediate HdaB specific adhesive functions.	Jul 2016
I04-1-Macromolecular Crystallography (fixed wavelength)	The extended structure of the periplasmic region of CdsD, a structural protein of the type III secretion system of Chlamydia trachomatis Gitte Meriläinen , M. Kristian Koski , Rik K. Wierenga Protein Science 25, 987 - 998 DOI: 10.1002/pro.2906 Show Abstract ▼ Abstract: The type III secretion system (T3SS) is required for the virulence of many gram-negative bacterial human pathogens. It is composed of several structural proteins, forming the secretion needle and its basis, the basal body. In Chlamydia spp., the T3SS inner membrane ring (IM-ring) of the basal body is formed by the periplasmic part of CdsD (outer ring) and CdsJ (inner ring). Here we describe the crystal structure of the C-terminal, periplasmic part of CdsD, not including the last 60 residues. Two crystal forms were obtained, grown in three different crystallization conditions. In both crystal forms there is one molecule per asymmetric unit adopting a similar extended structure. The structures consist of three periplasmic domains (PDs) of similar αββαβ topology as seen also in the structures of the homologous PrgH (Salmonella typhimurium) and YscD (Yersinia enterocolitica). Only in the C2 crystal form, there is a C-terminal additional helix after the PD3 domain. The relative orientation of the three subsequent CdsD PD domains with respect to each other is more extended than in PrgH but less extended than in YscD. Small-angle X-ray scattering data show that also in solution this CdsD construct adopts the same elongated shape. In both crystal forms the CdsD molecules are packed in a parallel fashion, using translational crystallographic symmetry. The most extensive crystal contacts are preserved in both crystal forms, suggesting a possible mode of assembly of the CdsD periplasmic part into a 24-mer complex forming the outer ring of the IM-ring of the T3SS.	May 2016
I24-Microfocus Macromolecular Crystallography	The crystal structure of NS5A domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric HCV inhibitors Sebastian Lambert , David R. Langley , James Garnett , Richard Angell , Katy Hedgethorne , Nicholas A. Meanwell , Steve J. Matthews Protein Science 23 (6), 723 - 734 DOI: 10.1002/pro.2456 PMID: 24639329 Diamond Proposal Number(s): [9424] Open Access Show Abstract ▼ Abstract: New direct acting antivirals (DAAs) such as daclatasvir (DCV; BMS-790052), which target NS5A function with picomolar potency, are showing promise in clinical trials. The exact nature of how these compounds have an inhibitory effect on HCV is unknown; however, major resistance mutations appear in the N-terminal region of NS5A that include the amphipathic helix and domain 1. The dimeric symmetry of these compounds suggests that they act on a dimer of NS5A, which is also consistent with the presence of dimers in crystals of NS5A domain 1 from genotype 1b. Genotype 1a HCV is less potently affected by these compounds and resistance mutations have a greater effect than in the 1b genotypes. We have obtained crystals of domain 1 of the important 1a NS5A homologue and intriguingly, our X-ray crystal structure reveals two new dimeric forms of this domain. Furthermore, the high solvent content (75%) makes it ideal for ligand-soaking. Daclatasvir (DCV) shows twofold symmetry suggesting NS5A dimers may be of physiological importance and serve as potential binding sites for DCV. These dimers also allow for new conformations of a NS5A expansive network which could explain its operation on the membranous web. Additionally, sulfates bound in the crystal structure may provide evidence for the previously proposed RNA binding groove, or explain regulation of NS5A domain 2 and 3 function and phosphorylation, by domain 1.	Jun 2014
I04-1-Macromolecular Crystallography (fixed wavelength)	Completing the structural family portrait of the human EphB tyrosine kinase domains Ross C. Overman , Judit E. Debreczeni , Caroline M. Truman , Mark S. Mcalister , Teresa K. Attwood Protein Science 23 (5), 627 - 638 DOI: 10.1002/pro.2445 PMID: 24677421 Show Abstract ▼ Abstract: The EphB receptors have key roles in cell morphology, adhesion, migration and invasion, and their aberrant action has been linked with the development and progression of many different tumor types. Their conflicting expression patterns in cancer tissues, combined with their high sequence and structural identity, present interesting challenges to those seeking to develop selective therapeutic molecules targeting this large receptor family. Here, we present the first structure of the EphB1 tyrosine kinase domain determined by X-ray crystallography to 2.5Å. Our comparative crystalisation analysis of the human EphB family kinases has also yielded new crystal forms of the human EphB2 and EphB4 catalytic domains. Unable to crystallize the wild-type EphB3 kinase domain, we used rational engineering (based on our new structures of EphB1, EphB2, and EphB4) to identify a single point mutation which facilitated its crystallization and structure determination to 2.2 Å. This mutation also improved the soluble recombinant yield of this kinase within Escherichia coli, and increased both its intrinsic stability and catalytic turnover, without affecting its ligand-binding profile. The partial ordering of the activation loop in the EphB3 structure alludes to a potential cis-phosphorylation mechanism for the EphB kinases. With the kinase domain structures of all four catalytically competent human EphB receptors now determined, a picture begins to emerge of possible opportunities to produce EphB isozyme-selective kinase inhibitors for mechanistic studies and therapeutic applications.	May 2014
I03-Macromolecular Crystallography	Structure of an early native-like intermediate of β2-microglobulin amyloidogenesis Saskia Vanderhaegen , Marcus Fislage , Katarzyna Domanska , Wim Versées , Els Pardon , Vittorio Bellotti , Jan Steyaert Protein Science 22 (10) DOI: 10.1002/pro.2321 PMID: 23904325 Open Access Show Abstract ▼ Abstract: To investigate early intermediates of β2-microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X-ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co-crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild-type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation.	Oct 2013

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